D. Tziafas, O. Pantelidou, A. Alvanou, G. Belibasakis & S. Papadimitriou
Departments of Endodontology, School of Dentistry, Histology and Embryology, Faculty of Medicine,
Clinical Sciences/Surgery Clinic, School of Veterinary Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece.
The objective of the present experiment was to study the early pulpal cell response and the onset of reparative dentine formation after capping application of MTA in mechanically exposed pulps.
The present experiments indicate that MTA is an effective pulp-capping material, able to stimulate hard tissue bridge formation during the early wound healing process. The stereotypic pulp defence mechanism by which primitive matrix (fibrodentine) trigger expression of the odontoblastic potential of pulpal cells seems to be related to the dentinogenic activity of MTA. Further experimental data are needed to determine whether interactions between extracellular matrix components (including fibronectin) and endogenous signaling molecules (growth factors) take place after pulp capping with MTA.
D. Tziafas, O. Pantelidou, A. Alvanou, G. Belibasakis & S. Papadimitriou
Departments of Endodontology, School of Dentistry, Histology and Embryology, Faculty of Medicine,
Clinical Sciences/Surgery Clinic, School of Veterinary Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece.
Direct pulp capping/pulpotomy is a well established method of treatment, in which the exposed dental pulp is covered with a suitable material that protects the pulp from additional injury and permits healing and repair (American Association of Endodontists 1981). Pulp capping is mainly indicated for reversible pulp tissue injury after physical or mechanical trauma on developing or mature teeth. Pulpotomy is also indicated for pulp exposures in carious developing teeth. The removal of the injurious challenge, the control of infection and the cytotoxic and biological properties of the capping material are critical factors influencing the treatment prognosis (Seltzer & Bender 1985). It is widely accepted that the ultimate goal of the application of a capping material is to induce the dentinogenic potential of pulpal cells (Schröder 1985, Stanley 1989, Mjör et al. 1991). The dentinogenic potential can be induced directly as a specific biological effect of the capping material on pulpal cells, or indirectly as a part of the stereotypic wound healing mechanism in the traumatized pulp (Baume 1980, Lesot et al. 1994).
Calcium hydroxide-based materials have found widespread use in traditional vital pulp therapy over a number of decades (Nyborg 1955, Tronstad 1974). The antibacterial effects of calcium hydroxide on infected pulp tissue are of considerable importance (Cox et al. 1982). Clinical and experimental observations in animal and human teeth have repeatedly demonstrated that reparative dentine formation is physiologically induced after pulp capping with calcium hydroxide (Nyborg 1955, Schröder & Granath 1971, Schröder & Sundström 1974, Fitzgerald 1979, Heys et al. 1990). The beneficial effect of Ca(OH) 2 -based materials is mainly attributed to the initial low-grade irritation of the traumatized pulp tissue, due to release of hydroxyl ions (Schröder 1985, Cvek et al. 1987). Calcium ions released from these materials and the subsequently formed anorganic precipitations (Holland et al. 1982, Hanks et al. 1983, Tziafas & Economides 1999) have recently been associated with the mechanism controlling cytological and functional changes in the interacting pulpal cells (Seux et al. 1991, Tziafas et al. 1995). The molecular interactions between pulpal extracellular matrix molecules and growth factors and their role as critical morphogenetic influences for initiation of reparative dentinogenesis after pulp capping with calcium hydroxide have been studied also (Lesot et al. 1993, Lesot et al. 1994, Yoshiba et al. 1996, Tziafas et al. 2000).
Pulp capping with calcium hydroxide remains an unpredictable method of treatment for mature teeth. A number of new agents have been tested during the last two decades as potential materials of choice (e.g. calcium phosphate ceramics, collagens, biologically active matrices and molecules, mineral trioxide aggregate (MTA). MTA has recently been introduced to clinical dentistry (Torabinejad & Chivian 1999). By culturing human osteosarcoma cells (Mitchell et al. 1999), or osteoblasts (Koh et al. 1997, Koh et al. 1998) in the presence of MTA, good cell growth was found. MTA further offered a biologically active substrate for cell attachment, whilst increased levels of alkaline phosphatase, osteocalcin, and interleukin-6 and -8 were measured. In vitro studies have demonstrated that the antibacterial effects of MTA are comparable to those of Ca(OH) 2 but it is more effective in preventing bacterial microleakage (Torabinejad et al. 1995). Pitt Ford et al. (1996) have documented that MTA placed in mechanically exposed pulps of monkeys stimulated pulp healing with minimal inflammatory reactions and dentinal bridge formation. The early pulpal cell response to MTA and the mechanism by which extracellular matrix is deposited after its application at pulp capping situations remains to be determined. The aim of the present study was to evaluate the early dentinogenic activity of MTA on mature dog teeth.
Materials and methods.
Three healthy dogs 12–18 months of age were used. All experimental procedures were carried out in accordance with European Communities Directive (86/609/EEC). The animals were anaesthetized with pentothal and intubated with a cuffed endotracheal tube at the beginning of the experimental procedures. Intact teeth (first molars, canines and third incisors from both jaws) with healthy periodontium were used. The teeth were isolated with cotton rolls, polished and washed with 0.5% chlorhexidine.
Buccal class V cavities were prepared by means of a size 31 inverted cone carbide bur in an air turbine with sterile saline spray. The pulps were exposed with a size 00 round bur in an air turbine, producing a wound the size of the cutting edge of the bur. The cavities were washed with sterile saline and dried with sterile cotton pellets. Light pressure with cotton pellets was applied to control haemorrhage.
The exposed pulps were capped with ProRoot MTA (Dentsply), which was mixed with sterile water to provide a grainy, sandy mixture. The material was placed at the exposure site and light pressure was applied with a wet cotton pellet according to the manufacturer’s instructions, resulting in cramming of material into the pulp space. The cavities were immediately restored with amalgam. Light pressure was applied to the amalgam during condensation. The teeth were treated sequentially so that three observation periods (1, 2 and 3 weeks) distributed amongst the three dogs were available. The animals were killed by an overdose of pentothal, the jaws were immediately dissected and the teeth were fixed and processed for light and electron microscopy analyses.
Twenty-one teeth distributed equally in the three observation periods were fixed in 10% buffered formalin (pH 7.2) and processed for light microscopic analysis. The teeth were demineralized for 40–50 days in 5% trichloroacetic acid and embedded in paraffin. Serial sections 6 m thick were cut labio-lingually through the exposure site and stained with Mayer’s haematoxylin-eosin and Gomori’s trichrome. The amalgam (with a part of the capping material) was gently removed before sectioning.
Six teeth, distributed equally in the 1- and 2-week observation periods, were processed for Transmission Electron Microscopic (TEM) analysis. Teeth were extracted and cut labio-lingually immediately after jaw’s dissection and fixed for 1 h in 3% glutaraldeyde buffered with 0.1 mol L –1 cacodylate (pH 7.3). The pulp tissue with the capping material was removed and small specimens of the capping area were fixed in the same fixative for 2 h. After rinsing in 0.1 mol L –1 cacodylate buffer, the samples were postfixed in 1% osmium-tetroxide in the same buffer and embedded in Epon 812. Semi-thin sections were stained with toluidine blue and ultrathin sections were stained with uranyl acetate and lead citrate.
Six teeth, distributed equally in the three observation periods, were processed for scanning electron microscopic (SEM) analysis. After fixation of the specimens in 10% buffered formalin, the pulp cavity of the teeth were cut and soft tissues were mechanically removed. The specimens were immersed in 5% sodium hypochlorite for 4 h and dehydrated in alcohol. The pulpal surface of the MTA were examined in a JEOL JSM-840 A SEM. In association with the SEM, X-ray microprobe analysis was performed for qualitative examination of the chemical composition of the MTA surface and the associated crystal deposits.
In all postoperative periods the light microscopy analysis showed histological changes in the superficial reaction zone close to the MTA–pulp interface. The underlying pulp tissue was consistently found to be of normal structure without any sign of inflammation or tissue degeneration. Only two 2-week specimens and one 3-week specimen showed haemorrhage in the central pulp core.
The reaction zone was of variable width and composed of pulpal cells, remnants of blood, areas of tissue coagulation, traces of capping material, dentine fragments, and postoperatively formed hard tissue. A few scattered inflammatory cells (predominantly macrophages and lymphocytes) were also seen in most of the examined specimens regardless of the observation period. The analysis was further focused at the reaction zone along the wound surface and the MTA–pulp interface.
Figure 1. TEM micrograph 7 days after application of MTA on mechanically exposed pulp of dog.
a) Pulpal cells arrangement along the exposed surface of the MTA and the associated crystalline structures. (x2500);
b) Higher magnification of 1a.
Numerous mitochondria and empty cisternae of the rough endoplasmic reticulum in the cytoplasm of the cell; direct contact between the cell surface and the crystals (arrows) distributed in the extracellular space of the reaction zone (x12000).
Figure 2. TEM micrograph 7 days after application of MTA on mechanically exposed pulp of dog. Numerous ribosomes and widened cisternae of the rough endoplasmic reticulum in the cytoplasm of the cell, which is in direct contact with free crystals (arrows) distributed in the extracellular space of the reaction zone (x20000).
Figure 3. TEM micrograph 7 days after application of MTA on mechanically exposed pulp of dog. Remnants of pulpal cells in close relation to the pulpal side of MTA ( 20 000).
One-week observation period.
An almost homogeneous front of organized crystalline structures or a thin zone of basophilic matrix was found along the pulpal side of MTA (Figs 1, 2). The TEM study showed elongated pulpal cells arranged at a distance 2–5 m from the crystalline front (Fig. 1a). These cells exhibited increased cytoplasmic/nucleus ratio, an eccentrically located nucleus and well developed cytoplasm with numerous mitochondria, free ribosomes, Golgi elements and rough endoplasmic reticulum (Figs 1b and 2). The apical pole of the elongated pulpal cells was associated with crystalline structures; cytoplasmic processes were also found in direct contact with the crystalline structures (Figs 1b and 2). Degenerated cells in contact with the capping material were also seen (Fig. 3).
The SEM study showed crystalline structures which ranged from intergrown crystal units to complicated oblong forms (Fig. 4a,b). The X-ray microanalysis of the examined surface (MTA and anorganic precipitations) revealed two high peaks corresponding to calcium and silicate and six low peaks corresponding to sodium, magnesium, aluminium, phosphorus, bismuth and iron (Fig. 4c). The oblong forms of crystals showed two peaks corresponding to calcium and phosphorus (Fig. 4d).
Figure 4. SEM micrographs (a and b) and associated X-ray analyses (c and d) 7 days after application of MTA on mechanically exposed pulp of dog. Non-homogenous crystal depositions onto the MTA surface
c) Two main peaks on MTA surface corresponding to calcium and silicate;
d) Two main peaks on crystalline structures corresponding to calcium and phosphorus.
Two-week observation period.
Production of hard tissue barrier consisting of irregular osteotypic matrix depositions with cellular inclusions was seen along the pulpal side of MTA in all teeth capped for 2 weeks (Fig. 5). The osteodentine barriers were found to be atubular in form and related to cuboidal or columnar cells.
Figure 5. Light microscopy micrograph 2 weeks after application of MTA on mechanically exposed pulp of dog. Osteodentinal matrix formation (arrows) along the pulpal side of MTA and haemorrhagic infiltration in the underlying pulp. (Hematoxylin-eosin, x40).
The TEM study showed a zone of densely packed collagen fibres elaborated in predentine-like pattern in direct contact with the front of electron dense crystalline-like structures (Fig. 6a). Spindle-shaped or tall columnar cells showing a well developed cytological organization with widened rough endoplasmic reticulum becoming parallel to the long axis of the cells were seen along the collagenous zone (Fig. 6b). Mitochondria, numerous Golgi saccules and dense bodies were distributed throughout the whole cytoplasm.
Figure 6. TEM micrograph 2 weeks after application of MTA on mechanically exposed pulp of dog.
a) New collagen matrix (NM) formation in direct contact with the zone of crystalline structures (CR) formed along the exposed surface of the MTA (x20000);
b) Numerous mitochondria and parallel cistrernae of the rough endoplasmic reticulum in the cytoplasm of the pulpal cells associated with the new collagenous matrix (NM) zone seen also in 6a (x8000).
The SEM study showed intergrown crystalline structures composed of rod-shaped or spherical units (Fig. 7). The X-ray microanalysis of the examined surface revealed two main peaks corresponding to calcium and phosphorus.
Three-week observation period.
Two-layered hard tissue barriers in contact with vital pulp completely bridging the pulpal wound surfaces were found in six out of seven specimens (Figs 8 and 9a). The barriers consisted of a coronal irregular layer of osteodentine and a pulpally tubular dentine-like matrix lined by elongated cells (Fig. 9b). In one specimen the bridge consisted only of an osteodentine layer lined in a number of serial sections by elongated and polarized cells (Fig. 10).
The pulpal surface of the barriers and the matrix deposited around the exposure site showed in SEM examination the typical surface structure of reparative dentine.
Many clinical and experimental studies have shown that mature dental pulp cells possess the ability to differentiate into a specific cell lineage forming tubular dentine in the absence of normal developmental conditions, i.e. dental epithelium and basement membrane (Baume 1980). This phenomenon takes place stereotypically, as an intrinsic defensive mechanism, in the repairing of pulp environment (Kakehashi et al. 1965, Yamamura 1985, Fitzgerald et al. 1990). The dentinogenic potential of dental ectomesenchymal cells is progressively expressed as a part of the natural healing process in the mature teeth (Baume 1980). It has been well recognized that dentinogenic activity can be observed in exposed pulps without any exogenous application (Kakehashi et al. 1965, Cox et al. 1987, Tsuji et al. 1987, Inoue & Shimono 1992). The superficial zone of extracellular matrix, which is stereotypically formed at the wound surface of the repairing connective tissues, is physiologically followed in exposed dental pulp by hard tissue deposition. A layer of pulp-specific formative cells (odontoblast-like cells) producing reparative dentine is finally formed as a sign of normalized pulp function. As odontoblast-like cells are recognized (in morphological terms), the elongated pulpal cells with increased cytoplasm/nucleus ratio and polarized nuclei, which form tubular matrix in a polar predentinelike pattern (Baume 1980, Lesot et al. 1994, Tziafas 1997).
Figure 7. SEM micrograph 2 weeks after application of MTA on mechanically exposed pulp of dog. Intergrown crystalline depositions onto the surface of MTA (x2000).
Figure 8. Light microscopy micrograph 3 weeks after application of MTA on mechanically exposed pulp of dog. Two-layered bridge formation consisting from osteodentine (arrows) and reparative dentine matrix (arrowheads) formation along the pulpal side of MTA (Hematoxylin-eosin, x40).
During the natural wound healing process in dental pulp, odontoblast-like cell differentiation and reparative dentine formation occurred in association with osteodentine or fibrodentine hard tissue formation (Baume 1980, Ruch 1985). This primitive type of pulp biomatrix seems to control the differentiation of pulpal cells into odontoblast-like cells and initiation of reparative dentine formation, substituting the dental epithelium and basement membrane to provide the necessary molecular influences (Ruch 1985, Lesot et al. 1994, Tziafas 1997). This mechanism controlling initiation of reparative dentinogenesis has been repeatedly confirmed after pulp capping with calcium hydroxide-based materials. Schröder (1985) reviewed the reparative process following capping of human teeth with calcium hydroxide: initially the cells under the wound surface proliferate, migrate and elaborate new collagen along the superficial necrotic zone or the pulpal surface of capping material. The necrotic zone and the new collagen layer attract mineral salts, becoming calcified matrices (fibrodentine). Then a layer of odontoblast-like cells is formed in association with the fibrodentine and reparative dentine is secreted. Many data from capping experiments suggest that initiation of reparative dentine formation might not be attributed to any specific dentinogenic effect of calcium hydroxide, although its effect in controlling infection and stimulating the wound healing process might not be excluded.
Figure 9. Light microscopy micrograph 3 weeks after application of MTA on mechanically exposed pulp of dog.
a) Two layered bridge formation consisting from osteodentin (arrow) and reparative dentin matrix (arrowheads) formation along the pulpal side of MTA (Hematoxylin-eosin, x40);
b) Higher magnification of 9a. Tubular matrix following osteodentin (arrow) associated with elongated formative cells (Hematoxylin-eosin, x200).
Figure 10. Light microscopy micrograph 3 weeks after application of MTA on mechanically exposed pulp of dog. Elongated polarized cells (arrowheads) associated with osteotypic matrix (arrows) formed in direct contact with the pulpal side of MTA (Hematoxylin-eosin, x200).
The present experiments demonstrated that pulp capping with MTA induces cytological and functional changes in pulpal cells, resulting in formation of fibrodentine and reparative dentine at the surface of mechanically exposed dental pulp. These observations are in agreement with those described previously in cultures of osteoblasts on MTA (Koh et al. 1997, Koh et al. 1998, Mitchell et al. 1999) or after application of MTA in capping situations (Pitt Ford et al. 1996). MTA offered a biologically active substrate for pulpal cells, able to regulate dentinogenic events. The pulp cell responses were studied here in short-term healing intervals to evaluate whether MTA induces directly reparative dentinogenesis, or the stereotypic intermediate formation of osteodentine/fibrodentine precedes any dentinogenic event. The initial effect of MTA on the surface of mechanically exposed pulp is the formation of a superficial layer of crystalline structures onto the pulpal surface of the capping material. Columnar cells undergoing nuclear and cytoplasmic polarization and showing a well developed cytoplasmic organization are further arranged along the crystalline structures. This short-term reaction clearly indicates stimulation of the biosynthetic activity of pulpal cells by the capping procedure but it could not be characterized as direct induction of reparative dentine formation. Initiation of reparative dentinogenesis can be identified in morphological studies only by the palisade appearance of elongated and polarized odontoblast- like cell layer able to secrete tubular matrix in a polar predentin-like pattern, although further biochemical and immuno-histochemical data are needed to characterize completely the nature and specificity of this phenomenon.
A new matrix of atubular form with cellular inclusions was observed beneath the capping material after 2 weeks. Elongated or cuboidal formative cells were found along this matrix. The TEM study showed collagen fibres, which had been densely packed in direct contact with the superficial crystalline layer. Formative cells associated with this matrix exhibited distinct organization throughout the whole cytoplasm, but no clear polarization. The SEM study of the pulpal side of this matrix showed an atypical surface structure and amorphous crystal precipitation. The data suggest a fibrodentinal nature of the newly synthesized matrix formed along the MTA–pulp interface (Baume 1980, Tziafas 1997).
Reparative dentinogenesis was clearly observed 3 weeks after capping of exposed pulps with MTA, in association with the firm fibrodentinal matrix. Odontoblastlike cells elaborating tubular matrix in a predentine-like pattern were seen in all cases. From the developmental point of view, these data confirm the similar mechanism for initiation of reparative dentinogenesis in capping with MTA and Ca(OH) 2 -based materials: in both agents capping of mechanically exposed pulps showed that fibrodentine matrix formation preceded any expression of the odontoblast-like cell phenotype (Schröder 1985, Fitzgerald et al. 1990). It seems that initiation of reparative dentine formation as a part of the natural wound healing process in MTA-treated pulps cannot be attributed to any specific dentino-inductive effect of the capping material. Nevertheless, the formation of an appropriate pulp environment due to the alkalinic properties of MTA favouring expression of the dentinogenic potential of pulpal cells, as has been suggested for Ca(OH) 2 -treated pulps (Torneck et al. 1983, Cvek et al. 1987), might be considered as a critical influence. The regulatory effect of MTA in production of osteocalcin or alkaline phosphatase, or interleukin-6 and -8 (Koh et al. 1997, Koh et al. 1998) might be further related to the stimulation of dentinogenic activity in the dental pulp treated with MTA. In addition, the important role of the fibronectin-rich zone, which is formed onto the crystalline structures (Seux et al. 1991, Tziafas et al. 1995, Yoshiba et al. 1996) observed along the pulpal side of MTA and a possible effect of the alkalinic environment in dissolution of growth factors from the surrounding dentine, as has been suggested for Ca(OH) 2 (Lesot et al. 1994), may not be excluded.
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