Dentinogenic potential of the dental pulp: facts and hypotheses

The aim of the present article is to discuss observations and hypotheses from different experimental approaches on the biological mechanisms underlying initiation of tertiary dentin formation and therapeutic control of pulp–dentinal regeneration. The specific dentinogenic potential of dental pulp cells in up‐regulating the biosynthetic activity of primary odontoblasts (reactionary dentinogenesis) and differentiation into odontoblast‐like cells (reparative dentinogenesis) is described. The role of biologically active matrices and molecules as signaling factors in the expression of the dentinogenic potential of dental pulp cells, in numerous ex vivo and in vivo models, is reviewed. Data are focused on the mechanisms by which the signaling molecules, in the presence of the appropriate pulp microenvironment and specific mechanical support, can induce competent pulpal cells in the acquisition of odontoblast‐like cell phenotype and reparative dentin formation. The ability of tissue engineering to stimulate reconstruction of the amputated pulp–dentin complex offers exciting opportunities for the future. Advances in molecular biology and bioengineering research might thus be integrated into the clinical problems of endodontology.
Received 13 February 2009; accepted 2 September 2009.     

Is hard tissue formation in the dental pulp after the death of the primary odontoblasts a regenerative or a reparative process?

Objectives

Conceptually, two types of tertiary dentine may be produced in response to caries and environmental irritations: “reactionary dentine” that is secreted by existing primary odontoblasts and “reparative dentine”, formed after the death of the odontoblasts by proliferation and differentiation of progenitor cells into odontoblast-like cells. Because histologic evidence for tubular dentine generated by newly differentiated odontoblast-like cells is lacking in human teeth, the present study examined pulpal cellular changes associated with caries/restorations, in the presence or absence of pulpal exposures.

Methods

Ninety-six extracted human teeth were histologically processed and serial sectioned for light microscopy: 65 contained untreated enamel/dentine caries; 20 were heavily restored and 11 had carious exposures managed by direct pulp-capping.

Results

Sparsely distributed, irregularly arranged dentinal tubules were identified from the tertiary dentine formed in teeth with unexposed medium/deep caries and in restored teeth; those tubules were continuous with the tubules of secondary dentine; in some cases, tubules were absent. The palisade odontoblast layer was reduced to a single layer of flattened cells. In direct pulp-capping of pulp exposures, the defects were repaired by the deposition of an amorphous dystrophic calcified tissue that resembled pulp stones more than dentine, sometimes entrapping pulpal remnants. This atubular hard tissue was lined by fibroblasts and collagen fibrils.

Conclusions

Histological evidence from the present study indicates that reparative dentinogenesis cannot be considered as a regenerative process since the so-formed hard tissue lacks tubular features characteristic of genuine dentine. Rather, this process represents a repair response that produces calcified scar tissues by pulpal fibroblasts.

Clinical significance

Formation of hard tissue in the dental pulp after the death of the primary odontoblasts has often been regarded by clinicians as regeneration of dentine. If the objective of the clinical procedures involved is to induce healing, reduce dentine hypersensitivity, or minimise future bacteria exposure, such procedures may be regarded as clinical success. However, current clinical treatment procedures are not adept at regenerating physiological dentne because the tissues formed in the dental pulp are more likely the result of repair responses via the formation of calcified scar tissues.     

Dental pulp regeneration via cell homing

The typical treatment for irreversibly inflamed/necrotic pulp tissue is root canal treatment. As an alternative approach, regenerative endodontics aims to regenerate dental pulp‐like tissues using two possible strategies: cell transplantation and cell homing. The former requires exogenously transplanted stem cells, complex procedures and high costs; the latter employs the host's endogenous cells to achieve tissue repair/regeneration, which is more clinically translatable. This systematic review examines cell homing for dental pulp regeneration, selecting articles on in vitro experiments, in vivo ectopic transplantation models and in situ pulp revascularization. MEDLINE/PubMed and Scopus databases were electronically searched for articles without limits in publication date. Two reviewers independently screened and included papers according to the predefined selection criteria. The electronic searches identified 46 studies. After title, abstract and full‐text examination, 10 articles met the inclusion criteria. In vitro data highlighted that multiple cytokines have the capacity to induce migration, proliferation and differentiation of dental pulp stem/progenitor cells. The majority of the in vivo studies obtained regenerated connective pulp‐like tissues with neovascularization. In some cases, the samples showed new innervation and new dentine deposition. The in situ pulp revascularization regenerated intracanal pulp‐like tissues with neovascularization, innervation and dentine formation. Cell homing strategies for pulp regeneration need further understanding and improvement if they are to become a reliable and effective approach in endodontics. Nevertheless, cell homing currently represents the most clinically viable pathway for dental pulp regeneration.     

Activation of human dental pulp progenitor/stem cells in response to odontoblast injury

In restorative dentistry, whilst moderate carious lesion treatment does not significantly compromise odontoblast cell survival, deep cavity preparation may lead to a partial death of these cells. However, newly formed odontoblast-like cells can replace the necrotic odontoblasts and secrete a reparative dentine matrix. Although several lines of evidence strongly suggest the presence of resting progenitor or stem cells in the dental pulp, little is known about the activation and migration of these cells in response to injury. Human immature third molars extracted for orthodontic reasons were used in this work to study the activation of progenitor/stem cells and their migration after deep cavity preparation involving in pulpal exposure using 5-bromo-2'-deoxyuridine labelling (BrdU). After incubation for 1 day, the BrdU was localised to the nuclei of cells in the perivascular area. The BrdU-immunolabelling exhibited a gradient. It was strong in the blood vessels surrounding the pulpal cavity and decreased in those away from the cavity. After incubation for 2 weeks, labelled cells were seen in the vicinity of the cavity. At 4 weeks, the immunolabelling was localised to the cavity area only. Control teeth without cavities or with shallow dentine cavities did not show any perivascular labelling after culture. These results clearly demonstrate that perivascular progenitor/stem cells can proliferate in response to odontoblast injury. They also show that these proliferating cells can migrate to the pulpal injury site in their tissue of origin simulating the situation in vivo.     

Tissue engineering in endodontics

The key elements of the regeneration of dentine-pulp complex are stem cells, morphogens and a scaffold of extracellular matrix. The pulp stem cells have the potential to differentiate into odontoblasts in response to bone morphogenetic proteins (BMPs). However, the use of BMPs in vivo has been restrained by lack of a suitable scaffold. Therefore, two alternative approaches, in vivo and ex vivo gene therapy were performed. Bmp I I/Gdf I I gene was directly transferred into amputated pulp by sonoporation and the reparative dentine formation was stimulated in vivo. However, there should be enough responsive stem cells in the pulp. Therefore, the isolated progenitor stem cells from pulp were transfected with Bmp I I/Gdf I I by electroporation and implanted onto the amputated pulp. This ex vivo gene therapy stimulated reparative dentine formation more optimally and rapidly compared with the in vivo gene therapy. These results suggest the possible clinical use of gene therapy of BMPs for endodontics.     

牙本质非胶原蛋白诱导牙髓-牙本质复合体反应的动物实验观察

目的：观察提取的猪牙本质非胶原蛋白对修复性牙本质形成的诱导活性。方法：用提取的猪EDTA可溶性牙本质非胶原蛋白作盖髓剂,对Wistar大鼠的健康第一磨牙进行直接盖髓实验,采用氢氧化钙（Dycal）和空白组为对照,通过组织学实验观察牙髓-牙本质复合体的反应。结果：盖髓术后7 d,各组之间软、硬组织反应的差异无统计学意义。术后14 d,NCPs组和Dycal组的修复反应均明显优于空白对照组（P〈0.01）,且NCPs组对牙髓的刺激小于Dycal组（P〈0.05）。结论：NCPs对牙髓刺激性小,可以诱导牙髓细胞分化为成牙本质细胞样细胞,形成修复性牙本质,其诱导活性至少与常规盖髓剂相当。     

层粘连蛋白在修复性牙本质形成中的免疫定位

目的 研究层粘连蛋白（laminin,LN）在牙髓修复中的免疫定位和分布特征。方法 在大鼠第一磨牙制备单面洞,分别观察3d、15d和30d后修复性牙本质的形成情况。以免疫组化技术检测LN的免疫反应。结果 术后3d,修复性牙本质尚未形成。牙髓内成牙本质细胞呈阳性染色,前期牙本质为弱阳性。术后15d,可见修复性牙本质形成和成牙本质细胞样细胞的出现,成牙本质细胞样细胞和牙髓细胞免疫染色呈阳性。术后30d,已形成的修复性牙本质内LN染色阳性,免疫活性特别集中于修复性牙本质与牙髓交界处,成牙本质细胞样细胞染色呈强阳性。结论 LN的分布特征提示,在修复性牙本质形成过程中,LN可能是牙髓细胞附着的一个有利因素。     

Odontoblast morphology and dental repair

Objectives. To investigate the changes in morphology and activity of pulp odontoblasts in response to cavity restoration variables and patient factors.Methods. Class V non exposed cavities were prepared in the intact 1st or 2nd premolar teeth of 27 patients, aged between 9 and 17 years-old. Following tooth extraction, the area of reactionary dentine and the area of the odontoblasts were measured using computerised histomorphometry.Results. The cytoplasm to nucleus ratio of the odontoblasts was found to increase beneath cut dentinal tubules, following the secretion of reactionary dentine. However, none of the patient or preparation variables were found to be correlated with changes in the odontoblast cytoplasm to nucleus ratio.Conclusions. Morphological changes in human odontoblasts is directly related to their capacity to repair dentine injuries and provide pulp protection. Changes in odontoblast morphology reflect secretory activity.     

再生性牙髓治疗方法的前景

再生性牙髓治疗是基于生物学基础治疗的一种方法,目的是替换受损的部分牙髓组织或允许牙髓样组织形成以完全替代原牙髓组织。针对牙髓炎或牙髓坏死的年轻恒牙的治疗,研究者们利用口腔来源干细胞或根尖周组织的干细胞建立各种利于修复的微环境,提出了如牙髓血运重建术/干细胞归巢治疗等充满前景的新治疗技术,从而为牙髓的再生提供了大量研究资料及可选治疗手段。该文对再生性牙髓治疗方法的现状及其发展前景作一综述。     

Stimulation of the rat dentine-pulp complex by bone morphogenetic protein-7 in vitro

Human recombinant bone morphogenetic protein-7 (BMP-7), when applied to freshly cut dentine in monkey teeth, stimulated tertiary dentine formation, but it is unclear whether this response involved upregulation of the synthetic and secretory activity of existing odontoblasts or the induction of differentiation of new odontoblast-like cells. Using a recently developed organ-culture system for whole tooth slices, the aim here was to examine the effects of BMP-7 on the stimulation and modulation of existing odontoblasts in the absence of tissue injury. Agarose beads were soaked in a 500 ng/ml or 100 ng/ml solution of BMP-7 in culture medium and placed on the odontoblast area of the dentine pulp complex of rat tooth slices. The slices were embedded in a semisolid agar-based medium and cultured at the liquid gas interface for 7 days. Results showed that beads soaked in 500 ng/ml BMP-7 stimulated a localized increase in extracellular matrix secretion by odontoblasts at the site of application, with greater stimulatory effects than from the lower concentration. These effects may be important in the reparative processes after tissue injury within the dentine-pulp complex and may be useful in the therapeutic induction of tertiary dentinogenesis.     

Comparison of pulp responses following restoration of exposed and non-exposed cavities

OBJECTIVES: The purpose of this study is to compare and contrast differences of pulp responses between non-exposed and exposed cavity preparations in terms of inflammation, frequency of bacterial microleakage, odontoblast and odontoblastoid cell numbers, and tertiary dentine formation. METHODS: Class V non-exposed cavities (n=161) and exposed cavities (n=161 teeth) were prepared in non-human primate teeth. Cavities were restored with calcium hydroxide [Ca(OH)(2)], resin modified glass ionomer, or resin composite. Following extraction (7-730 days), bacteria were detected with McKays stain and pulp reactions were categorized according to ISO guidelines. Teeth were analyzed histomorphometrically and statistically using analysis of variance tests. RESULTS: Exposed cavities in comparison with non-exposed cavities were found to have more severe inflammation (p=0.0001), greater quantities of tertiary dentine (p=0.0001), and an increased frequency of bacterial microleakage (p=0.0034). The density of odontoblastoid cells beneath pulp exposed tertiary dentine was found to be 47.8% of odontoblast cell density beneath non-exposed dentine (p=0.0001). CONCLUSIONS: The restoration of exposed cavity preparations is associated with more traumatic pulp injury and repair responses. Consequently, efforts should be made to minimize iatrogenic dentine removal during cavity preparation and the creation of pulp exposures whenever possible.     

Stem cell‐based pulp tissue engineering: variables enrolled in translation from the bench to the bedside,a systematic review of literature

Stem cell‐based therapy (SC‐BT) is emerging as an alternative for endodontic therapies. The interaction between stem cells and scaffolds plays a crucial role in the generation of a ‘friendly cell’ microenvironment. The aim of this systematic review was to explore techniques applied to regenerate the pulp–dentine complex tissue using SC‐BT. An electronic search into the SciVerse Scopus (SS), ISI Web Science (IWS) and Entrez PubMed (EP) using specific keywords was performed. Specific inclusion and exclusion criteria were predetermined. The search yielded papers, out of which full‐text papers were included in the final analyses. Data extraction pooled the results in four main topics: (a) influence of the chemical properties of the scaffolds over cell behaviour; (b) influence of the physical characteristics of scaffolds over cell behaviour; (c) strategies applied to improve the stem cell/scaffold interface; and (d) influence of cue microenvironment on stem cell differentiation towards odontoblast‐like cells and pulp‐like tissue formation. The relationship between the scaffolds, the environment and the growth factors released from dentine are critical for de novo pulp tissue regeneration. The preconditioning of dentine walls with ethylenediaminetetraacetic acid (EDTA) was imperative for successful pulp–dentine complex regeneration. An analyses of the grouped results revealed that pulp regeneration was an attainable goal.     

The effect of cavity restoration variables on odontoblast cell numbers and dental repair

OBJECTIVES: Dentinal repair following cavity restoration is dependent on several parameters including the numbers of surviving odontoblasts. The purpose of this study was to examine the effects of cavity cutting and restoration treatments on post-operative odontoblast numbers. METHODS: 353 Standardised non-exposed rectangular Class V cavities, were cut into the buccal dentin of intact 1st or 2nd premolar teeth of 165 patients, aged between nine and 25 years of age. Composite cavity restorations with various etching treatments were compared with resin-modified glass ionomer cements, enamel bonding resins, as well as polycarboxylate, calcium hydroxide, and zinc oxide eugenol materials. Following tooth extraction (20-381 days) for orthodontic reasons, the area of the reactionary dentine and the area of the odontoblasts was measured histomorphometrically. RESULTS: Odontoblast numbers and dentine repair activity were found to be influenced more by cavity restoration variables, than the choice of cavity filling materials or patient factors. The most important cavity preparation variable was the cavity remaining dentine thickness (RDT); below 0.25mm the numbers of odontoblasts decreased by 23%, and minimal reactionary dentine repair was observed. CONCLUSIONS: Odontoblast injury increased as the cavity RDT decreased. In rank order of maintaining odontoblast numbers beneath restored cavities with a RDT below 0.5mm, and using calcium hydroxide for comparison; calcium hydroxide (100%), polycarboxylate (82.4%), zinc oxide eugenol (81.3%), composite (75.5%), enamel bonding resin (49.5%) and RMGIC (42.8%). The vitality and dentine repair capacity of the pulp is dependent on odontoblast survival. Variations in the extent of odontoblast injury caused during operative procedures, may be the major underlying reason for the success or failure of restorative treatments.     

Primary molar pulp therapy--histological evaluation of failure

Objectives. Qualitative comparison of the clinical, radiographic and histological outcomes in a small sample of cariously exposed primary molars, extracted after unsucessful pulp treatment by two vital pulpotomy methods. Subjects and methods. The primary molars were extracted, because of treatment failure, during a longitudinal clinical investigation of the relative efficacy of 20% Buckley’s Formocresol versus calcium hydroxide powder as pulp dressings in vital pulp therapy (in press). Fifty‐two child patients were sequentially enrolled in the longitudinal clinical investigation, 26 boys and 26 girls. Primary molar teeth requiring vital pulp therapy were randomly allocated to either the formocresol group (F) or the calcium hydroxide group (C). Coronal pulp amputation was prescribed only in teeth with vital, cariously‐exposed pulp tissue. All cases were reviewed using predefined clinical and radiological criteria. Seventy‐nine cariously‐exposed primary molars required vital pulp therapy. Forty‐four teeth were included in group F and 35 in group C. Five per cent (n = 2) of teeth in group F and 11 per cent of teeth (n = 4) in group C were terminated from the trial due to clinical and/or radiographic failure. Of the six teeth extracted, five were sufficiently intact to be retained for histological evaluation. Results. Post‐extraction radiographs taken before specimen preparation showed reactionary dentine barrier (bridge) formation in teeth treated with calcium hydroxide. However, the narrowing of root canals, indicative of appositional reactionary dentine deposition, was seen in both groups (F and C). Histological examination confirmed these findings and revealed that pus cells were evident in all specimens examined. There was also histological evidence of resorption of reactionary dentine within the root canal and that forming the calcified barrier (dentine bridge). Conclusion. The clinical and radiographic outcomes for the five teeth correlate well with the reported findings from decalcified histological section. Although numbers are small, the histological findings may indicate possible reasons for treatment failure. There are few reports of this sort in the literature of this under‐researched treatment modality.     

Pulp biology: progress during the past 25 years

During the past 25 years there has been a rapid expansion in our knowledge of the dentine and pulp complex. This paper provides representative examples of important advances that researchers have made in this field. Topics to be considered include: differentiation of odontoblasts, dentine matrix proteins, extent of odontoblast processes, pulpal stem cells, apoptosis, interstitial fluid pressure in normal and inflamed pulps, class II antigen-presenting cells of the pulp, cytokines, antibodies, pulpal calcifications, tertiary dentine and pulpal inflammation associated with bacterial contamination of exposed dentine beneath restorations.     

Preserving the vital pulp in operative dentistry: 3. Thickness of remaining cavity dentine as a key mediator of pulpal injury and repair responses

Confusion surrounds the pivotal role played by the remaining dentine thickness in a cavity in determining pulpal injury and repair response outcomes after restorative treatment. The multifactorial nature of the injury repair response requires that attention is focused on the most important factors, including remaining dentine thickness, to harness the natural regenerative properties of the pulp and to avoid postoperative treatment complications.     

Transdentinal stimulation of reactionary dentinogenesis in ferrets by dentine matrix components

OBJECTIVES: Trans-dentinal stimulation of reactionary dentinogenesis may be mediated through cellular signalling by bio-active components released from the dentine matrix during injury. Understanding of these processes will be important to guide dentinal repair activity following restorative surgery. The purpose of this study was to investigate the effects of implanting isolated dentine matrix proteins within cavity preparations on dentinal repair activity and odontoblast survival using a controlled experimental animal model. METHODS: Forty-five ferret canine teeth each had a standardised non-exposed cylindrical Class V cavity cut into the buccal dentine. Ten cavities were restored with zinc oxide eugenol, as a control. Two different lyophilised preparations of dentine matrix components were implanted on the axial floor of the remaining 35 cavities prior to filling with zinc oxide eugenol. After post-operative periods of 2, 7, 14, 28, and 90 days, the teeth were extracted and examined histomorphometrically and the data analysed statistically by analysis of variance tests. RESULTS: The odontoblasts beneath the restored cavities responded to the presence of the two dentine matrix preparations by increasing the mean area of reactionary dentine secreted by 433 and 578%, and the numbers of odontoblasts remained stable. CONCLUSION: Dentine matrix components can stimulate reactionary dentinogenesis in non-exposed cavity preparations. It will now be important to identify how this may be harnessed as a part of routine restorative surgery to optimise treatment outcomes with a biological basis.     

牙髓根尖周病的治疗新方法：再生牙髓病学之路

为促进牙髓牙本质复合体的再生,学者们将组织工程学技术应用于牙髓治疗中,并提出了再生牙髓病学的概念。作为口腔医学的新兴学科,再生牙髓病学运用牙源性干细胞、生物支架以及生长因子等因素促进牙根继续发育和牙髓组织再生。临床上,牙髓再生技术已应用于治疗年轻恒牙牙髓坏死及根尖周炎,越来越多的病例报道或研究结果显示牙髓再生的可能性,牙髓再生治疗将成为口腔医生治疗牙髓病的新选择。本文就再生牙髓病学的现状和发展进行总结分析。     

The influence of haemostatic agents on healing of healthy human dental pulp tissue capped with calcium hydroxide

AIM: To investigate the hypothesis that different haemostatic agents could impair the histological response of human pulps capped with calcium hydroxide. METHODOLOGY: Forty-five third molars scheduled for extraction were selected. Class I cavities with pulp exposures were prepared. Three agents were used to control bleeding: 0.9% saline solution (control, n = 14), 5.25% sodium hypochlorite (n = 16) and 2% chlorhexidine digluconate (n = 15). The pulps were dressed with hard-setting calcium hydroxide cement. After 7, 30 or 90 days, teeth were extracted, formalin-fixed and prepared for histochemical techniques. The biological response was categorized using the following criteria: inflammatory response, soft tissue organization, reactionary dentine and reparative dentine. Data were submitted to statistical analysis, using nonparametric Kruskal-Wallis one-way analysis of variance on ranks. Differences amongst groups were detected using Dunn's method. RESULTS: The statistical analysis disclosed that whilst inflammatory response decreased over time, reactionary dentine deposition and reparative dentine formation increased in the latter periods of evaluation (P < 0.05). The three agents had similar performances for all criteria evaluated. The conventional pulp response to calcium hydroxide was observed over time, and complete pulp healing was observed in 88% of the specimens after 90 days. CONCLUSION: The three haemostatic agents did not impair the healing process following pulp exposure and capping with calcium hydroxide at different time intervals investigated.     

干细胞和支架与牙髓再生及其血运重建

在牙髓再生中,获取干细胞的方法包括干细胞移植、细胞归巢和诱导出血。干细胞移植可产生异位的牙髓样组织,可控制移植细胞的数量并选择对牙髓再生潜在效能最佳的细胞亚种。细胞归巢是指利用信号分子招募宿主内源性干细胞至需治疗的牙体根管中增殖和分化,形成牙髓-牙本质样组织。诱导根尖出血进入根管为年轻恒牙牙髓再生的一个重要步骤。支架是细胞在合成组织时的支撑结构,可促进细胞黏附,为牙髓再生提供有利的环境。牙髓再生离不开血运重建或者血管再生,感染控制、根管预处理、冠方封闭等操作,可为牙髓再生包括其血运重建提供适宜的环境。总之,组织工程技术在牙髓领域的应用发展为牙髓再生带来了新的希望。