Clin Oral Invest DOI 10.1007/s00784-015-1555-8
ORIGINAL ARTICLE
Regenerative endodontic procedure of an infected immature permanent human tooth: an immunohistological study Nastaran Meschi 1 & Petra Hilkens 2 & Ivo Lambrichts 2 & Kathleen Van den Eynde 3 & Athina Mavridou 1 & Olaf Strijbos 1 & Marieke De Ketelaere 4 & Gertrude Van Gorp 1 & Paul Lambrechts 1
Received: 24 January 2015 / Accepted: 28 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Objectives An immunohistological study of an infected immature permanent human tooth after a regenerative endodontic procedure (REP) was conducted in order to determine the histologic outcome of this procedure. Besides observed signs of angiogenesis and neurogenesis, repair and/or regeneration of the pulp-dentin complex was also investigated. Materials and methods A REP was performed on tooth 45 of a 10-year-old girl. Eleven months post-treatment, the tooth had to be removed for orthodontic reasons. The following investigations were performed: immunohistology and radiographic quantification of root development. After hematoxylin-eosin (HE) staining, the following immunomarkers were selected: neurofilament (NF), pan cytokeratin (PK), osteocalcin (OC), and CD34. Results The REP resulted in clinical and radiographic healing of the periradicular lesion and quantifiable root development. The HE staining matches with the medical imaging post-REP: underneath the mineral trioxide aggregate a calcified bridge with cell inclusions, connective pulp-like tissue (PLT) Electronic supplementary material The online version of this article (doi:10.1007/s00784-015-1555-8) contains supplementary material, which is available to authorized users. * Nastaran Meschi [email protected] 1
Department of Oral Health Sciences, KU Leuven & Dentistry, University Hospitals Leuven, Leuven, Belgium
2
Laboratory of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, 3590 Diepenbeek, Belgium
3
Laboratory of Translational Cell and Tissue Research, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium
4
H. Horriestraat 40 a bus 13, 8800 Roeselare, Belgium
with blood vessels, osteodentin against the root canal walls, on the root surface cementum (Ce), and periodontal ligament (PDL). The PDL was PK+. The blood vessels in the PLT and PDL were CD34+. The Ce, osteodentin, and stromal cells in the PLT were OC+. The neurovascular bundles in the PLT were NF+. Conclusions Immunohistologically, REP of this infected immature permanent tooth resulted in an intracanalar connective tissue with a regulated physiology, but not pulp tissue. Clinical relevance REP of an immature permanent infected tooth may heal the periapical infection and may result in a combination of regeneration and repair of the pulp-dentin complex. Keywords Angiogenesis . Pulp biology . Stem cell(s) . Tooth development
Introduction Over the last few decades, a number of treatment procedures have been described to treat necrotic or inflamed immature permanent teeth in order to achieve apexogenesis and maturogenesis so that the tooth and the alveolar bone can be preserved at least until full skeletal growth. While different terms have been used for these procedures, such as revascularization, revitalization, and regenerative endodontic procedures (REPs) [1, 2], the concept has remained the same. Huang [3] mentioned this concept as an alternative for apexification and distinguished two outcomes: ingrowth of periodontal tissue (repair) or regeneration of the pulp-dentin complex. In 1961, Nygaard-Ostby [4, 5] was one of the first to describe the histological nature of the newly formed tissue in the root canal of infected teeth post-REP. The findings of his animal and human studies showed new fibrous tissue
Clin Oral Invest
formation, root canal wall resorption, and deposition of cementum-like tissue. Decades later, animal histological studies after REP of infected immature teeth [6–9] reported a reparative process as well: periodontal and hard tissue ingrowth into the root canal. Even though pulp-like tissue (PLT) was detected in root canals of human teeth in cases with follow-up after REP of less than 2 years [8, 10, 11] (Table 2), the histological reports of human teeth with at least 24 months of follow-up [12–14] concur with the animal studies, independent of the REP method used (classic revascularization as described by Banchs and Trope [15] or with platelet-rich plasma as scaffold). One of the main topics of the International Association for Dental Research Symposium of the Pulp Biology and Regeneration Group in 2013 was the assessment of the newly formed tissue inside the root canal after REP [16]. Thus, the purpose of the present study was to determine the histological outcome of a REP by means of a thorough immunohistological study of an infected immature permanent human tooth 11 months post-REP. The dental pulp consists of highly vascularized and innervated connective tissue, and its regenerative capacity is due to the presence of mesenchymal stem cells [17, 18]. To that end, we sought for evidence of angiogenesis, neurogenesis, and repair and/or regeneration of the pulp-dentin complex. Until now, to our knowledge, no study on infected human immature permanent teeth less than 1 year post-REP has been reported yet, in which the presence of blood vessels, nervous tissue, and Hertwig’s epithelial root sheath (HERS) has been immunohistologically investigated (Table 2).
Materials and methods Clinical procedure Due to an acute apical abscess on tooth 45 of a 10-year-old Asian girl, the general practitioner prescribed antibiotics and referred her to the endodontist. The endodontist observed an occlusal carious fissure and an immature root with a periradicular lesion (Fig. 1a). The patient’s orthodontist recommended that the tooth be preserved for 1 more year, so a REP was performed. At the first session, the endodontist administered a local anesthetic, isolated the tooth, and disinfected the root canal with 1 % sodium hypochlorite at 45 °C. The root canal was then dried with paper points. To prevent tooth discoloration, a double antibiotic paste (DAP) without minocycline (ciprofloxacin 200 mg, metronidazole 500 mg, macrogel ointment, and propylene glycol) [19] was injected into the root canal, and the tooth was covered with a glass ionomer temporary filling (Fuji II LC). A week later, the swelling became worse, and unfortunately, a general practitioner left the tooth in open drainage. The endodontist
repeated the treatment procedure of the first session. During the third and last session, the endodontist disinfected the root canal again with 1 % sodium hypochlorite at 45 °C, dried it with paper points, and rinsed it with 17 % EDTA. Afterwards, bleeding was induced into the root canal by triggering the periapex with a K-file .40. Subsequently, Hemocol™ (Medical Biomaterial Products, GmbH), a sterile hemostatic plug made of porcine collagen, was placed on the blood clot to serve as a scaffold for mineral trioxide aggregate (MTA, ProRoot®). The endodontist filled the tooth crown with glass ionomer cement and composite (Fig. 1b). Furthermore, the general practitioner was asked to seal the coronal fissure of the contralateral premolar. Eleven months after REP (Fig. 1c), the orthodontist referred the patient to an oral and maxillofacial surgeon to remove the first maxillary premolars and the second mandibular premolars under general anesthesia. Two months after the extraction, we received a radiograph of the region of the extracted right mandibular second premolar (Fig. 1f) from the orthodontist. Tissue processing and immunohistochemical procedures REP tooth Immediately after tooth 45 was extracted, it was fixed in glutaraldehyde for a week. Cone beam CT (J Morita® Accuitomo) and micro-CT (Skyscan 1172®, reconstructed to video with VG Studio Max 2.0®) images were created of the tooth in the fixation fluid (Fig. 1d, e). After 3 months of immersion in EDTA with polyvinyl pyrrolidone (10 % EDTA in 0.1 M Tris buffer, pH 6.95; polyvinyl pyrrolidone 7.5 %), the tooth was demineralized enough for histology (checked radiographically). We stored the tooth in 0.2 M cacodylic acid sodium salt trihydrate, until sectioning. The tooth was sliced longitudinally into two parts, to improve penetration of the paraffin. Processing was done overnight by an Excelsior TP device: The sample was dehydrated in solutions of methanol with increasing concentration and subsequently prepared for embedding in paraffin by solutions of toluene with increasing concentration. Both pieces were then imbedded in paraffin. After hardening of the paraffin, the coronal composite was drilled out with water cooling under a microscope (Fig. 1g) in order to prevent blunting of the sectioning blade. Subsequently, longitudinal serial sections (per 5 μm; Fig. 1h) were made. After hematoxylin-eosin (HE) staining, the following immunohistochemical markers were chosen: CD34, neurofilament (NF), pan cytokeratin (PK), and osteocalcin (OC). The autostainer BOND® MAX (Leica®) performed the immunoh i s t o c h e m i c a l s t a i n i n g ( Ta b l e 1 ) . B r i e f l y, a f t e r deparaffinization, the PK and NF slices were pretreated with Tris/EDTA at pH 9 for 20 min to obtain antigen retrieval. For
Clin Oral Invest
Fig. 1 The REP tooth. a A part of the panoramic radiograph showing an immature permanent right lower second premolar with apical periodontitis; b periapical radiograph after REP; c periapical radiograph 11 months after REP; d CBCT saggital view post-extraction; e CBCT
transversal view post-extraction; f periapical radiograph of the alveolar bone 2 months after extraction of the REP tooth; g longitudinally sectioned, embedded in paraffin, and after removal of the coronal composite and glass ionomer cement; h serial sections
the other antibodies, this pretreatment was not performed because the risk of damaging the sections was deemed to be too high. The peroxidase block was then executed, and the sections were incubated with the primary antibodies NF, CO, PK, and CD34 (rabbit anti-mouse) for 30 min and then for 8 min with the secondary (goat anti-rabbit) antibodies. The sections were then washed in Wash Buffer (BOND® Wash Solution 10x Concentrate) and incubated with a horseradishperoxidase-labeled polymer (goat anti-rabbit; BOND® polymer refine detection kit) for 8 min. After immersing in diaminobenzidine for 10 min, the slices were counterstained for 5 min with hematoxylin. The slices were inspected under light microscope (Nikon® Eclipse 80i) and scanned with the Mirax Desk (Zeiss®).
Quantification of root development The periapical radiographs of the last REP session (Fig. 1b) and 11 months after REP (Fig. 1c) were aligned with the ImageJ plug-in tool TurboReg (US National Institutes of Health, Bethesda, MD) following the procedure described by Bose et al. [21]. Subsequently, the changes in root length and width were assessed with the ImageJ software, following the method described by Shimizu et al. [13].
Results REP tooth
Control tooth
Hematoxylin-eosin staining
For comparison, histology of a human tooth 11 with a Bnormal pulp^ has been performed, following the protocol (from fixation to image processing of the slices) described by Mavridou et al. [20]. The staining was performed with Stevenel’s blue and Von Gieson’s picrofuchsin, visualizing mineralized tissue (red) and non-mineralized tissue (blue).
The histological overview (Fig. 2a) matches the radiographs (Fig. 1b–e and micro-CT (see Supplementary materials)). From coronal to apical, the following is visible: MTA, a calcified bridge (CB) with cell inclusions (Fig. 2a, f), connective pulp-like tissue (PLT) with blood vessels (Fig. 2d), and most apically, blood plasma (BP).
Table 1 The primary antibodies: manufacturer, clone, dilution. and positive control
Antibody
Manufacturer
Clone
Dilution
Positive control
NF OC PK CD34
Dako® R&D Systems® Dako® Dako®
2F11 190125 AE1/AE3 Qbend 10
1/500 1/750 RTU RTU
Tongue: reactive epithelial hyperplasia Osteoblastoma Tongue: reactive epithelial hyperplasia –
NF neurofilament, OC osteocalcin, PK pan cytokeratin, RTU ready to use
Clin Oral Invest
Fig. 2 Hematoxylin-eosin- and CD34-stained longitudinal sections of the REP tooth. a An overview of the REP tooth from coronal to apical: MTA, calcified bridge (CB), pulp-like tissue (PLT), blood plasma (BP); b on the external root surface cementum (Ce) underneath the periodontal ligament (PDL), the arrow points to external root resorption and subsequent cementum (Ce) apposition; c osteodentin (OD) deposition on the inner wall of the root dentin (rD), the arrow points to internal
root resorption; d the arrows point to blood vessels in the PLT; e blood vessel stained brown by CD34; f magnification of the CB with cell inclusions (see arrows). Figure 2a, b, c, and f was selected from the images scanned by the Mirax Desk (Zeiss®). The other figures were selected during light microscopic inspection. Scale bars: a 1000 μm; b 200 μm; c 100 μm; d–f 50 μm
Internal resorption of the root dentin and osteodentin apposition (atubular tertiary dentine with cells entrapped) [21] are noted on the internal dentin walls (Fig. 2c). External root surface resorption and cementum (Ce) apposition (Fig. 2b) appear underneath the periodontal ligament (PDL).
Control tooth
Immunohistochemical staining The sections were CD34+ with the blood vessels stained brown (Fig. 2e). The stromal cells in the PLT reacted positive to the OC staining (Fig. 3a–c). Particularly, the cementoblasts against the Ce and the osteodentin (OD) producing cells against the OD were OC+ (Fig. 3c, d). Figure 3d presents inactive cementocytes in the Ce, with increasing diameters toward the PDL. A similar image occurs in the OD (Fig. 3c). The PDL did not react significantly with the OC marker. For the PK staining, only the PDL was PK+: some epithelial rests of Malassez (ERM) stained brown (Fig. 3e, see arrow). But, the PLT was totally PK−. Furthermore, the sections were NF+ since there are several neurovascular bundles (NVBs, Fig. 4c–e). A typical image of a sympathetic nerve fiber is shown in Fig. 4a, b.
Figure 5 presents the axial histological slices of a Bnormal^ human tooth. The vascularized pulp (P) signals the odontoblasts (Od) to secrete unmineralized predentin (PD), which is mineralized afterward and becomes root dentin (rD). Radiographic evaluation post-REP The REP led to radiographical healing of the periradicular lesion of tooth 45 (Fig. 1b, c). On the radiograph taken by the orthodontist 2 months post-extraction (Fig. 1f), the radioopaque lumen of the former root canal of tooth 45 may suggest the presence of calcified tissue in the root canal. Quantification of root development In order to prevent false positive results due to the presence of a CB, the root width and canal width assessments at midroot level (as described by Shimizu et al. [13]) were excluded. At the 11-month recall, we assessed a mean increase of 9.5 % in root length and a mean increase of 29.1 % in root width. These findings are in accordance with the overall findings concerning the increase in root width and length post-REP:
Clin Oral Invest Fig. 3 Immunohistochemical stained slides of the REP tooth with osteocalcin (OC) (a–d) and pan cytokeratin (PK) (e). a An overview of an OC-stained slide showing OC+ pulp-like tissue (PLT) and a calcified bridge (CB) underneath the MTA; b, c the PLT and osteodentin (OD) are OC+; d the root cementum (Ce) is OC+; e epithelial rests of Malassez in the periodontal ligament (PDL) is PK+ (see arrow). Figure 3a is selected from the images scanned by the Mirax Desk (Zeiss®). The other figures were selected with light microscopic inspection. Scale bars: a 500 μm; b, c 50 μm; d, e 20 μm
an increase of 30 % in root width and length is expected respectively at 1 and 3 years post-REP [2, 21].
Discussion The purpose of this immunohistological study was to search for evidence of angiogenesis, neurogenesis, and repair and/or regeneration of the pulp-dentin complex in an infected immature human tooth 11 months post-REP. CD34 was selected as an immunomarker for vascularization [22, 23]. Not only the HE-stained sections but also the immunostaining showed evidence of angiogenesis (Fig. 2d–f). Nevertheless, the apical Fig. 4 Immunohistochemical stained slides of the REP tooth with neurofilament (NF). a A sympathetic neuron (attached to a blood vessel, see arrow on Fig. 4b) in the pulp-like tissue (PLT); b a magnified view of Fig. 4a; c an overview of a neurovascular bundle (NVB) in the PLT (CB = calcified bridge); d magnification of the NVB on Fig. 4c; e magnification of the blue selected area of Fig. 4d. Scale bars: a, d 50 μm; b, e 20 μm; c 100 μm
blood plasma (Fig. 2a) was caused by bleeding after extraction. Similarities may be observed between the vascularized PLT of the REP tooth and the vascularized pulp of the normal tooth (Fig. 5). In order to investigate neurogenesis, the cytoskeletal protein NF was chosen as marker for nerve fibers, because it can stain unmyelinated nerve tissue [23–25]. Although a typical image of a sympathetic nerve fiber in relation to blood vessels has been shown (Fig. 4a, b), the positive staining for NF does not reveal the precise nature of the nerve fibers. Tooth development is based on an interaction between epithelial and mesenchymal cells. HERS directs the root development [26] and finally breaks down in the epithelial rests of
Clin Oral Invest Fig. 5 Histological axial sections of a human tooth 11 with a Bnormal pulp.^ a, b Od odontoblasts, PD predentin, rD root dentin, P pulp; arrows point to blood vessels in the pulp; scale bars: a 100 μm; b 20 μm
Malassez (ERM) [23], for which PK was chosen as marker in this report. However, the PK findings show evidence of the cessation of physiological root maturation. Obviously, one can find apically no remnants of HERS since an ERM is PK+ in the PDL (Fig. 3e). It has been suggested that the cessation of HERS growth is a cement-inductive signal [25]. No HERS also implies no signaling to the apical papilla for further root dentin formation. Nevertheless, in half of the histological studies on human teeth post-REP reported until now (Table 2), the direct [10] or indirect [13, 14] presence of HERS has been described. However, none of these studies has performed an (additional) immunohistological staining to confirm the presence of HERS. At last OC, was selected as a marker for osteoblasts and odontoblasts in demineralized tissue [23, 27]. More specifically for teeth, OC is a non-collagenous protein of the dentinal extracellular matrix [18]. It also measures the viability of human dental pulp [28, 29]. The viability of the fibrotic PLT is thus shown by its OC+ staining. However, in order to detect new odontoblast formation, more important dentin-specific
Table 2
markers such as dentin sialoprotein and nestin [27, 30] should have been used. As shown by the immunohistochemical staining, despite the infection, a viable connective tissue was created in the root canal post-REP. The infection of the tooth was caused by caries in a pre-existing deep occlusal fissure although it was not an Oehler’s invagination type. Limited to no instrumentation is advised in REPs [2] although the question still remains if enough disinfection is obtained [11, 31]. Except for one histological report of a human tooth, all other studies mentioned in Table 2 were performed on infected teeth. Absence of infection would enhance the chance of survival of stem cells and auto-revascularization [5, 31]; thus, the histological outcome of non-infected teeth would be more in favor of regeneration. Notwithstanding the acute apical abscess in the present report, we can match the radiographical bone healing (Fig. 1c) to the histological findings inside the tooth, as there is a normal dispersion of inflammatory cells in the PLT (Fig. 2a, d). Nevertheless, the aggression of the infection
Human histological studies investigating the nature of tissue formed in the root canal post–regenerative endodontic procedure (REP)
fist author (year)
Infected (I)/ non-infected (NI)
PRP
REP follow-up
Blood vessels (HE)
Nervous tissue
HERS (HE)
Pulp-like tissue
Intracanalar calcification
Shimizu et al. (2012) [10] Torabinejad et al. (2012) [8] Martin et al. (2013) [12] Shimizu et al. (2013) [13] Becerra et al. (2014) [14] Lin et al. (2014) [11]
NI I I I I I
− + + − − −
3.5 weeks 14 months 25 months 26 months 24 months 16 months
+ + + − + −
n.s. n.s. n.s. − n.s. n.s.
+ n.a. − + + −
+ + − − + −
− − + + + +
All studies reported the histology of one single tooth post-REP HE hematoxylin-eosin staining, PRP platelet-rich plasma, HERS Hertwig’s epithelial root sheath, n.s. no staining performed, n.a. non-applicable due to the fact that only the intracanal tissue was investigated and not the entire tooth
Clin Oral Invest
marked the external root surface (Fig. 2b): external root resorption and subsequent cementum apposition are visible. A similar image is seen at the inner dentinal walls (Fig. 2c), but then dentinoclasts have probably resorbed the dentin [7]. This clastic activity can also release growth factors for further repair or regeneration of the dentin, as is the case with the formation of OD as described in an external cervical resorption report [32]. The cellular inclusions in the outer Ce and inner OD also show similarities (Fig. 3d, c): from vital cells toward the PLT or PDL and cells with reduced metabolic activity at the outer margin. Cellular Ce is mostly found in the apical root, its function being to maintain the tooth in occlusion and to repair diseased Ce (as in dentin). These cementocytes embedded in Ce possess a canalicular network that makes intercommunication possible as well as communication with the cementoblasts at the PDL interface [18]. This is also the case for the OD: vital cells imbedded in the OD can maintain the continuous OD apposition and cause further calcification of the root canal space, which leads to root thickening, as also assessed by the radiographical quantification. Histologically, we cannot prove root lengthening in this case because the tooth was fixed without the surrounding periapical tissue and the root tip could have been damaged during extraction. But, a significant root lengthening could radiographically be measured. Nevertheless, due to the fact that the HERS was not functional as mentioned above, the assessed root development was caused by a reparative process. This is also obvious when the histology of the normal tooth (Fig. 5) is compared to that of the REP tooth (Fig. 2): the Od and PD layer, both crucial for continuous physiological mineralization of the rD, are missing in the REP tooth. Regardless of the CB, which is induced by the osteogenic activity of the MTA [33], several factors show reparative osteogenic and cementogenic activity inside the root canal: the similarities between OD and Ce, the potential for further OD apposition (as mentioned above), the OC+ PLT, and the periapical radiograph 2 months post-extraction (Fig. 1f). Even if the overall results are more in accordance with the short-term histological studies [8, 10, 11], these findings imply that, if the tooth were extracted later, the histology would probably be similar to the animal [6–9] and human long-term histological reports (Table 2): more calcified tissue inside the root canal. One explanation for these findings is that, because a blood clot was created by triggering the periapical tissue, pre-osteoblasts and pre-cementoblasts of the bone marrow and PDL might proliferate and migrate into the wounded area [9]. Another reason could be the DAP concentration. A capsule of ciprofloxacin 200 mg and one of metronidazole 500 mg were mixed with macrogel ointment and propylene glycol. As the amount of gel applied was arbitrary, the exact concentration of the DAP injected in the root canal was unfortunately not known. Thus, if there had been any vital pulp and/or stem cells despite the periapical infection, the applied concentration
of DAP might have been lethal for these cells so that regeneration would have been limited [2, 34, 35]. To summarize, in this report, the REP did not induce pulp tissue, but a true connective tissue with a regulated physiology: fibroblasts surrounded by nerve fibers and blood vessels. However, the REP was in this report successful as there was radiographical healing of the periradicular lesion and a quantifiable root development. The ideal outcome of a REP would be de novo regeneration of the pulp-dentin complex. Despite the clinical simplicity of the existing treatment protocols, this Bideal outcome^ in humans has yet to be achieved (Table 2). Furthermore, the question remains if the newly formed reparative tissue in the root canal prevents the tooth from fracturing, as there is no evidence available about the fracture resistance of this tissue. More robustly designed investigations are necessary in this field of endodontology, as the contribution of case reports in the level of evidence is limited. Regarding the tissue engineering attempts, cell-based approaches are more promising than cell-free ones. However, there are some challenges, among them being the unfortunate lack of recognition of the medical field of cell-based regenerative endodontic therapy [36].
Conclusion The REP led to clinical and radiographical healing of the periapical infection and an increased root width and length. Immunohistologically, this treatment resulted in a combination of regeneration and repair of the pulp-dentin complex. Compliance with ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. This article does not contain any studies with animals performed by any of the authors. Cnflict of interest The authors declare that they have no competing interests.
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ORIGINAL ARTICLE
Regenerative endodontic procedure of an infected immature permanent human tooth: an immunohistological study Nastaran Meschi 1 & Petra Hilkens 2 & Ivo Lambrichts 2 & Kathleen Van den Eynde 3 & Athina Mavridou 1 & Olaf Strijbos 1 & Marieke De Ketelaere 4 & Gertrude Van Gorp 1 & Paul Lambrechts 1
Received: 24 January 2015 / Accepted: 28 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Objectives An immunohistological study of an infected immature permanent human tooth after a regenerative endodontic procedure (REP) was conducted in order to determine the histologic outcome of this procedure. Besides observed signs of angiogenesis and neurogenesis, repair and/or regeneration of the pulp-dentin complex was also investigated. Materials and methods A REP was performed on tooth 45 of a 10-year-old girl. Eleven months post-treatment, the tooth had to be removed for orthodontic reasons. The following investigations were performed: immunohistology and radiographic quantification of root development. After hematoxylin-eosin (HE) staining, the following immunomarkers were selected: neurofilament (NF), pan cytokeratin (PK), osteocalcin (OC), and CD34. Results The REP resulted in clinical and radiographic healing of the periradicular lesion and quantifiable root development. The HE staining matches with the medical imaging post-REP: underneath the mineral trioxide aggregate a calcified bridge with cell inclusions, connective pulp-like tissue (PLT) Electronic supplementary material The online version of this article (doi:10.1007/s00784-015-1555-8) contains supplementary material, which is available to authorized users. * Nastaran Meschi [email protected] 1
Department of Oral Health Sciences, KU Leuven & Dentistry, University Hospitals Leuven, Leuven, Belgium
2
Laboratory of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, 3590 Diepenbeek, Belgium
3
Laboratory of Translational Cell and Tissue Research, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium
4
H. Horriestraat 40 a bus 13, 8800 Roeselare, Belgium
with blood vessels, osteodentin against the root canal walls, on the root surface cementum (Ce), and periodontal ligament (PDL). The PDL was PK+. The blood vessels in the PLT and PDL were CD34+. The Ce, osteodentin, and stromal cells in the PLT were OC+. The neurovascular bundles in the PLT were NF+. Conclusions Immunohistologically, REP of this infected immature permanent tooth resulted in an intracanalar connective tissue with a regulated physiology, but not pulp tissue. Clinical relevance REP of an immature permanent infected tooth may heal the periapical infection and may result in a combination of regeneration and repair of the pulp-dentin complex. Keywords Angiogenesis . Pulp biology . Stem cell(s) . Tooth development
Introduction Over the last few decades, a number of treatment procedures have been described to treat necrotic or inflamed immature permanent teeth in order to achieve apexogenesis and maturogenesis so that the tooth and the alveolar bone can be preserved at least until full skeletal growth. While different terms have been used for these procedures, such as revascularization, revitalization, and regenerative endodontic procedures (REPs) [1, 2], the concept has remained the same. Huang [3] mentioned this concept as an alternative for apexification and distinguished two outcomes: ingrowth of periodontal tissue (repair) or regeneration of the pulp-dentin complex. In 1961, Nygaard-Ostby [4, 5] was one of the first to describe the histological nature of the newly formed tissue in the root canal of infected teeth post-REP. The findings of his animal and human studies showed new fibrous tissue
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formation, root canal wall resorption, and deposition of cementum-like tissue. Decades later, animal histological studies after REP of infected immature teeth [6–9] reported a reparative process as well: periodontal and hard tissue ingrowth into the root canal. Even though pulp-like tissue (PLT) was detected in root canals of human teeth in cases with follow-up after REP of less than 2 years [8, 10, 11] (Table 2), the histological reports of human teeth with at least 24 months of follow-up [12–14] concur with the animal studies, independent of the REP method used (classic revascularization as described by Banchs and Trope [15] or with platelet-rich plasma as scaffold). One of the main topics of the International Association for Dental Research Symposium of the Pulp Biology and Regeneration Group in 2013 was the assessment of the newly formed tissue inside the root canal after REP [16]. Thus, the purpose of the present study was to determine the histological outcome of a REP by means of a thorough immunohistological study of an infected immature permanent human tooth 11 months post-REP. The dental pulp consists of highly vascularized and innervated connective tissue, and its regenerative capacity is due to the presence of mesenchymal stem cells [17, 18]. To that end, we sought for evidence of angiogenesis, neurogenesis, and repair and/or regeneration of the pulp-dentin complex. Until now, to our knowledge, no study on infected human immature permanent teeth less than 1 year post-REP has been reported yet, in which the presence of blood vessels, nervous tissue, and Hertwig’s epithelial root sheath (HERS) has been immunohistologically investigated (Table 2).
Materials and methods Clinical procedure Due to an acute apical abscess on tooth 45 of a 10-year-old Asian girl, the general practitioner prescribed antibiotics and referred her to the endodontist. The endodontist observed an occlusal carious fissure and an immature root with a periradicular lesion (Fig. 1a). The patient’s orthodontist recommended that the tooth be preserved for 1 more year, so a REP was performed. At the first session, the endodontist administered a local anesthetic, isolated the tooth, and disinfected the root canal with 1 % sodium hypochlorite at 45 °C. The root canal was then dried with paper points. To prevent tooth discoloration, a double antibiotic paste (DAP) without minocycline (ciprofloxacin 200 mg, metronidazole 500 mg, macrogel ointment, and propylene glycol) [19] was injected into the root canal, and the tooth was covered with a glass ionomer temporary filling (Fuji II LC). A week later, the swelling became worse, and unfortunately, a general practitioner left the tooth in open drainage. The endodontist
repeated the treatment procedure of the first session. During the third and last session, the endodontist disinfected the root canal again with 1 % sodium hypochlorite at 45 °C, dried it with paper points, and rinsed it with 17 % EDTA. Afterwards, bleeding was induced into the root canal by triggering the periapex with a K-file .40. Subsequently, Hemocol™ (Medical Biomaterial Products, GmbH), a sterile hemostatic plug made of porcine collagen, was placed on the blood clot to serve as a scaffold for mineral trioxide aggregate (MTA, ProRoot®). The endodontist filled the tooth crown with glass ionomer cement and composite (Fig. 1b). Furthermore, the general practitioner was asked to seal the coronal fissure of the contralateral premolar. Eleven months after REP (Fig. 1c), the orthodontist referred the patient to an oral and maxillofacial surgeon to remove the first maxillary premolars and the second mandibular premolars under general anesthesia. Two months after the extraction, we received a radiograph of the region of the extracted right mandibular second premolar (Fig. 1f) from the orthodontist. Tissue processing and immunohistochemical procedures REP tooth Immediately after tooth 45 was extracted, it was fixed in glutaraldehyde for a week. Cone beam CT (J Morita® Accuitomo) and micro-CT (Skyscan 1172®, reconstructed to video with VG Studio Max 2.0®) images were created of the tooth in the fixation fluid (Fig. 1d, e). After 3 months of immersion in EDTA with polyvinyl pyrrolidone (10 % EDTA in 0.1 M Tris buffer, pH 6.95; polyvinyl pyrrolidone 7.5 %), the tooth was demineralized enough for histology (checked radiographically). We stored the tooth in 0.2 M cacodylic acid sodium salt trihydrate, until sectioning. The tooth was sliced longitudinally into two parts, to improve penetration of the paraffin. Processing was done overnight by an Excelsior TP device: The sample was dehydrated in solutions of methanol with increasing concentration and subsequently prepared for embedding in paraffin by solutions of toluene with increasing concentration. Both pieces were then imbedded in paraffin. After hardening of the paraffin, the coronal composite was drilled out with water cooling under a microscope (Fig. 1g) in order to prevent blunting of the sectioning blade. Subsequently, longitudinal serial sections (per 5 μm; Fig. 1h) were made. After hematoxylin-eosin (HE) staining, the following immunohistochemical markers were chosen: CD34, neurofilament (NF), pan cytokeratin (PK), and osteocalcin (OC). The autostainer BOND® MAX (Leica®) performed the immunoh i s t o c h e m i c a l s t a i n i n g ( Ta b l e 1 ) . B r i e f l y, a f t e r deparaffinization, the PK and NF slices were pretreated with Tris/EDTA at pH 9 for 20 min to obtain antigen retrieval. For
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Fig. 1 The REP tooth. a A part of the panoramic radiograph showing an immature permanent right lower second premolar with apical periodontitis; b periapical radiograph after REP; c periapical radiograph 11 months after REP; d CBCT saggital view post-extraction; e CBCT
transversal view post-extraction; f periapical radiograph of the alveolar bone 2 months after extraction of the REP tooth; g longitudinally sectioned, embedded in paraffin, and after removal of the coronal composite and glass ionomer cement; h serial sections
the other antibodies, this pretreatment was not performed because the risk of damaging the sections was deemed to be too high. The peroxidase block was then executed, and the sections were incubated with the primary antibodies NF, CO, PK, and CD34 (rabbit anti-mouse) for 30 min and then for 8 min with the secondary (goat anti-rabbit) antibodies. The sections were then washed in Wash Buffer (BOND® Wash Solution 10x Concentrate) and incubated with a horseradishperoxidase-labeled polymer (goat anti-rabbit; BOND® polymer refine detection kit) for 8 min. After immersing in diaminobenzidine for 10 min, the slices were counterstained for 5 min with hematoxylin. The slices were inspected under light microscope (Nikon® Eclipse 80i) and scanned with the Mirax Desk (Zeiss®).
Quantification of root development The periapical radiographs of the last REP session (Fig. 1b) and 11 months after REP (Fig. 1c) were aligned with the ImageJ plug-in tool TurboReg (US National Institutes of Health, Bethesda, MD) following the procedure described by Bose et al. [21]. Subsequently, the changes in root length and width were assessed with the ImageJ software, following the method described by Shimizu et al. [13].
Results REP tooth
Control tooth
Hematoxylin-eosin staining
For comparison, histology of a human tooth 11 with a Bnormal pulp^ has been performed, following the protocol (from fixation to image processing of the slices) described by Mavridou et al. [20]. The staining was performed with Stevenel’s blue and Von Gieson’s picrofuchsin, visualizing mineralized tissue (red) and non-mineralized tissue (blue).
The histological overview (Fig. 2a) matches the radiographs (Fig. 1b–e and micro-CT (see Supplementary materials)). From coronal to apical, the following is visible: MTA, a calcified bridge (CB) with cell inclusions (Fig. 2a, f), connective pulp-like tissue (PLT) with blood vessels (Fig. 2d), and most apically, blood plasma (BP).
Table 1 The primary antibodies: manufacturer, clone, dilution. and positive control
Antibody
Manufacturer
Clone
Dilution
Positive control
NF OC PK CD34
Dako® R&D Systems® Dako® Dako®
2F11 190125 AE1/AE3 Qbend 10
1/500 1/750 RTU RTU
Tongue: reactive epithelial hyperplasia Osteoblastoma Tongue: reactive epithelial hyperplasia –
NF neurofilament, OC osteocalcin, PK pan cytokeratin, RTU ready to use
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Fig. 2 Hematoxylin-eosin- and CD34-stained longitudinal sections of the REP tooth. a An overview of the REP tooth from coronal to apical: MTA, calcified bridge (CB), pulp-like tissue (PLT), blood plasma (BP); b on the external root surface cementum (Ce) underneath the periodontal ligament (PDL), the arrow points to external root resorption and subsequent cementum (Ce) apposition; c osteodentin (OD) deposition on the inner wall of the root dentin (rD), the arrow points to internal
root resorption; d the arrows point to blood vessels in the PLT; e blood vessel stained brown by CD34; f magnification of the CB with cell inclusions (see arrows). Figure 2a, b, c, and f was selected from the images scanned by the Mirax Desk (Zeiss®). The other figures were selected during light microscopic inspection. Scale bars: a 1000 μm; b 200 μm; c 100 μm; d–f 50 μm
Internal resorption of the root dentin and osteodentin apposition (atubular tertiary dentine with cells entrapped) [21] are noted on the internal dentin walls (Fig. 2c). External root surface resorption and cementum (Ce) apposition (Fig. 2b) appear underneath the periodontal ligament (PDL).
Control tooth
Immunohistochemical staining The sections were CD34+ with the blood vessels stained brown (Fig. 2e). The stromal cells in the PLT reacted positive to the OC staining (Fig. 3a–c). Particularly, the cementoblasts against the Ce and the osteodentin (OD) producing cells against the OD were OC+ (Fig. 3c, d). Figure 3d presents inactive cementocytes in the Ce, with increasing diameters toward the PDL. A similar image occurs in the OD (Fig. 3c). The PDL did not react significantly with the OC marker. For the PK staining, only the PDL was PK+: some epithelial rests of Malassez (ERM) stained brown (Fig. 3e, see arrow). But, the PLT was totally PK−. Furthermore, the sections were NF+ since there are several neurovascular bundles (NVBs, Fig. 4c–e). A typical image of a sympathetic nerve fiber is shown in Fig. 4a, b.
Figure 5 presents the axial histological slices of a Bnormal^ human tooth. The vascularized pulp (P) signals the odontoblasts (Od) to secrete unmineralized predentin (PD), which is mineralized afterward and becomes root dentin (rD). Radiographic evaluation post-REP The REP led to radiographical healing of the periradicular lesion of tooth 45 (Fig. 1b, c). On the radiograph taken by the orthodontist 2 months post-extraction (Fig. 1f), the radioopaque lumen of the former root canal of tooth 45 may suggest the presence of calcified tissue in the root canal. Quantification of root development In order to prevent false positive results due to the presence of a CB, the root width and canal width assessments at midroot level (as described by Shimizu et al. [13]) were excluded. At the 11-month recall, we assessed a mean increase of 9.5 % in root length and a mean increase of 29.1 % in root width. These findings are in accordance with the overall findings concerning the increase in root width and length post-REP:
Clin Oral Invest Fig. 3 Immunohistochemical stained slides of the REP tooth with osteocalcin (OC) (a–d) and pan cytokeratin (PK) (e). a An overview of an OC-stained slide showing OC+ pulp-like tissue (PLT) and a calcified bridge (CB) underneath the MTA; b, c the PLT and osteodentin (OD) are OC+; d the root cementum (Ce) is OC+; e epithelial rests of Malassez in the periodontal ligament (PDL) is PK+ (see arrow). Figure 3a is selected from the images scanned by the Mirax Desk (Zeiss®). The other figures were selected with light microscopic inspection. Scale bars: a 500 μm; b, c 50 μm; d, e 20 μm
an increase of 30 % in root width and length is expected respectively at 1 and 3 years post-REP [2, 21].
Discussion The purpose of this immunohistological study was to search for evidence of angiogenesis, neurogenesis, and repair and/or regeneration of the pulp-dentin complex in an infected immature human tooth 11 months post-REP. CD34 was selected as an immunomarker for vascularization [22, 23]. Not only the HE-stained sections but also the immunostaining showed evidence of angiogenesis (Fig. 2d–f). Nevertheless, the apical Fig. 4 Immunohistochemical stained slides of the REP tooth with neurofilament (NF). a A sympathetic neuron (attached to a blood vessel, see arrow on Fig. 4b) in the pulp-like tissue (PLT); b a magnified view of Fig. 4a; c an overview of a neurovascular bundle (NVB) in the PLT (CB = calcified bridge); d magnification of the NVB on Fig. 4c; e magnification of the blue selected area of Fig. 4d. Scale bars: a, d 50 μm; b, e 20 μm; c 100 μm
blood plasma (Fig. 2a) was caused by bleeding after extraction. Similarities may be observed between the vascularized PLT of the REP tooth and the vascularized pulp of the normal tooth (Fig. 5). In order to investigate neurogenesis, the cytoskeletal protein NF was chosen as marker for nerve fibers, because it can stain unmyelinated nerve tissue [23–25]. Although a typical image of a sympathetic nerve fiber in relation to blood vessels has been shown (Fig. 4a, b), the positive staining for NF does not reveal the precise nature of the nerve fibers. Tooth development is based on an interaction between epithelial and mesenchymal cells. HERS directs the root development [26] and finally breaks down in the epithelial rests of
Clin Oral Invest Fig. 5 Histological axial sections of a human tooth 11 with a Bnormal pulp.^ a, b Od odontoblasts, PD predentin, rD root dentin, P pulp; arrows point to blood vessels in the pulp; scale bars: a 100 μm; b 20 μm
Malassez (ERM) [23], for which PK was chosen as marker in this report. However, the PK findings show evidence of the cessation of physiological root maturation. Obviously, one can find apically no remnants of HERS since an ERM is PK+ in the PDL (Fig. 3e). It has been suggested that the cessation of HERS growth is a cement-inductive signal [25]. No HERS also implies no signaling to the apical papilla for further root dentin formation. Nevertheless, in half of the histological studies on human teeth post-REP reported until now (Table 2), the direct [10] or indirect [13, 14] presence of HERS has been described. However, none of these studies has performed an (additional) immunohistological staining to confirm the presence of HERS. At last OC, was selected as a marker for osteoblasts and odontoblasts in demineralized tissue [23, 27]. More specifically for teeth, OC is a non-collagenous protein of the dentinal extracellular matrix [18]. It also measures the viability of human dental pulp [28, 29]. The viability of the fibrotic PLT is thus shown by its OC+ staining. However, in order to detect new odontoblast formation, more important dentin-specific
Table 2
markers such as dentin sialoprotein and nestin [27, 30] should have been used. As shown by the immunohistochemical staining, despite the infection, a viable connective tissue was created in the root canal post-REP. The infection of the tooth was caused by caries in a pre-existing deep occlusal fissure although it was not an Oehler’s invagination type. Limited to no instrumentation is advised in REPs [2] although the question still remains if enough disinfection is obtained [11, 31]. Except for one histological report of a human tooth, all other studies mentioned in Table 2 were performed on infected teeth. Absence of infection would enhance the chance of survival of stem cells and auto-revascularization [5, 31]; thus, the histological outcome of non-infected teeth would be more in favor of regeneration. Notwithstanding the acute apical abscess in the present report, we can match the radiographical bone healing (Fig. 1c) to the histological findings inside the tooth, as there is a normal dispersion of inflammatory cells in the PLT (Fig. 2a, d). Nevertheless, the aggression of the infection
Human histological studies investigating the nature of tissue formed in the root canal post–regenerative endodontic procedure (REP)
fist author (year)
Infected (I)/ non-infected (NI)
PRP
REP follow-up
Blood vessels (HE)
Nervous tissue
HERS (HE)
Pulp-like tissue
Intracanalar calcification
Shimizu et al. (2012) [10] Torabinejad et al. (2012) [8] Martin et al. (2013) [12] Shimizu et al. (2013) [13] Becerra et al. (2014) [14] Lin et al. (2014) [11]
NI I I I I I
− + + − − −
3.5 weeks 14 months 25 months 26 months 24 months 16 months
+ + + − + −
n.s. n.s. n.s. − n.s. n.s.
+ n.a. − + + −
+ + − − + −
− − + + + +
All studies reported the histology of one single tooth post-REP HE hematoxylin-eosin staining, PRP platelet-rich plasma, HERS Hertwig’s epithelial root sheath, n.s. no staining performed, n.a. non-applicable due to the fact that only the intracanal tissue was investigated and not the entire tooth
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marked the external root surface (Fig. 2b): external root resorption and subsequent cementum apposition are visible. A similar image is seen at the inner dentinal walls (Fig. 2c), but then dentinoclasts have probably resorbed the dentin [7]. This clastic activity can also release growth factors for further repair or regeneration of the dentin, as is the case with the formation of OD as described in an external cervical resorption report [32]. The cellular inclusions in the outer Ce and inner OD also show similarities (Fig. 3d, c): from vital cells toward the PLT or PDL and cells with reduced metabolic activity at the outer margin. Cellular Ce is mostly found in the apical root, its function being to maintain the tooth in occlusion and to repair diseased Ce (as in dentin). These cementocytes embedded in Ce possess a canalicular network that makes intercommunication possible as well as communication with the cementoblasts at the PDL interface [18]. This is also the case for the OD: vital cells imbedded in the OD can maintain the continuous OD apposition and cause further calcification of the root canal space, which leads to root thickening, as also assessed by the radiographical quantification. Histologically, we cannot prove root lengthening in this case because the tooth was fixed without the surrounding periapical tissue and the root tip could have been damaged during extraction. But, a significant root lengthening could radiographically be measured. Nevertheless, due to the fact that the HERS was not functional as mentioned above, the assessed root development was caused by a reparative process. This is also obvious when the histology of the normal tooth (Fig. 5) is compared to that of the REP tooth (Fig. 2): the Od and PD layer, both crucial for continuous physiological mineralization of the rD, are missing in the REP tooth. Regardless of the CB, which is induced by the osteogenic activity of the MTA [33], several factors show reparative osteogenic and cementogenic activity inside the root canal: the similarities between OD and Ce, the potential for further OD apposition (as mentioned above), the OC+ PLT, and the periapical radiograph 2 months post-extraction (Fig. 1f). Even if the overall results are more in accordance with the short-term histological studies [8, 10, 11], these findings imply that, if the tooth were extracted later, the histology would probably be similar to the animal [6–9] and human long-term histological reports (Table 2): more calcified tissue inside the root canal. One explanation for these findings is that, because a blood clot was created by triggering the periapical tissue, pre-osteoblasts and pre-cementoblasts of the bone marrow and PDL might proliferate and migrate into the wounded area [9]. Another reason could be the DAP concentration. A capsule of ciprofloxacin 200 mg and one of metronidazole 500 mg were mixed with macrogel ointment and propylene glycol. As the amount of gel applied was arbitrary, the exact concentration of the DAP injected in the root canal was unfortunately not known. Thus, if there had been any vital pulp and/or stem cells despite the periapical infection, the applied concentration
of DAP might have been lethal for these cells so that regeneration would have been limited [2, 34, 35]. To summarize, in this report, the REP did not induce pulp tissue, but a true connective tissue with a regulated physiology: fibroblasts surrounded by nerve fibers and blood vessels. However, the REP was in this report successful as there was radiographical healing of the periradicular lesion and a quantifiable root development. The ideal outcome of a REP would be de novo regeneration of the pulp-dentin complex. Despite the clinical simplicity of the existing treatment protocols, this Bideal outcome^ in humans has yet to be achieved (Table 2). Furthermore, the question remains if the newly formed reparative tissue in the root canal prevents the tooth from fracturing, as there is no evidence available about the fracture resistance of this tissue. More robustly designed investigations are necessary in this field of endodontology, as the contribution of case reports in the level of evidence is limited. Regarding the tissue engineering attempts, cell-based approaches are more promising than cell-free ones. However, there are some challenges, among them being the unfortunate lack of recognition of the medical field of cell-based regenerative endodontic therapy [36].
Conclusion The REP led to clinical and radiographical healing of the periapical infection and an increased root width and length. Immunohistologically, this treatment resulted in a combination of regeneration and repair of the pulp-dentin complex. Compliance with ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. This article does not contain any studies with animals performed by any of the authors. Cnflict of interest The authors declare that they have no competing interests.
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