Basic Research—Technology
Scanning Electron Microscopic Study on the Fibrillar Structures within Dentinal Tubules of Human Dentin Maricela Garces-Ortız, DDS, MSc, PhD,* Constantino Ledesma-Montes, DDS, MSc, PhD,* and Jose Reyes-Gasga, MSc, PhD† Abstract Introduction: Pulp biology is central to the whole tooth, and knowledge on its microstructure is changing with new studies. This study presents certain microfibrillar structures found within the dentin tubules of human teeth connecting dentin tubules and odontoblastic processes. Methods: We analyzed the crowns of 30 noncarious, human teeth. They were fixed; demineralized; and, later, processed and reviewed by means of scanning electron microscopy. Results: In the predentin layer, we found numerous fine fibrillar structures connecting the odontoblastic process and the wall of the dentinal tubule. In the inner dentinal third, we observed structures forming a dense microfibrillar network of variable thickness and diameters. These microstructures were very thin and numerous in this area, and their number decreased as more external dentin levels were examined. Conclusions: According to the review of the literature and our findings, these microfibrillar structures may be an unrecognized support system that holds and secures the odontoblastic process within the dentinal tubule (J Endod 2015;41:1510–1514)
Key Words Dentin, dentinal tubules, microfibrils, micromembranes, odontoblastic process
From the *Clinical Oral Pathology Laboratory, Facultad de Odontologıa and †New Materials Laboratory, Instituto de Fısica, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico. Address requests for reprints to Dr Constantino LedesmaMontes, Clinical Oral Pathology Laboratory, Division de Estudios de Posgrado e Investigacion, Facultad de Odontologıa, Universidad Nacional Autonoma de Mexico, Mexico, 04510 DF Mexico. E-mail address:
[email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.02.026
D
entin is a mineralized connective tissue with a self-recovering capacity. Dentin is a complex structure composed by odontoblasts, their odontoblastic processes (OPs), dentin tubules (DTs), noncollagenous proteins, and mineralized collagen forming the main dentinal corps. Dentin is classified into the following 3 types: primary, secondary, and tertiary. Only the primary and secondary dentin forms the dentin tissue of the normal noncarious teeth. DTs enclose and protect OPs from environmental harmful stimuli, and OPs secrete the proteic matrix formed by collagen and noncollagen proteins mainly developing in a mineralizing protein complex (1). Odontoblasts are dentin-forming cells; they are tall columnar cells located in the frontier between pulp tissue and primary dentin. Their functions include secretion of the dentin matrix proteins; they are responsible for the mineral deposition process (2); and they are involved in the transmission of stimuli from the external environment to the pulp, helping in sensitivity to painful stimuli (3, 4). Since the first description by Tomes (5), the odontoblastic process is not a simple cytoplasmic extension of the odontoblast; it is located within the DT and secretes all the dentin proteic and nonproteic components involved in the dentinal biomineralization process. There are several structures composing the dentin, and the OP is an extremely complex biologic odontoblastic structure because of the cellular polarization; it is located within the DT and reaches different distances at different dentin levels. The peritubular dentin surrounds this important structure, and, finally, the intertubular dentin encloses both structures (6–16). Some researchers stated they found OPs located in the predentin zone only (7). Others reported the presence of OPs within the first third of the dentin thickness (6, 8–11), and in other studies, OPs were observed in the outer third reaching the dentinoenamel junction (10, 12–15). Fox et al (16) studied OPs in the root of extracted human third molars and found they were in contact with the dentinocemental junction. Interestingly, in teeth from Macaca mulatta monkeys, Kelley et al (13) observed that OPs were present in both the inner and outer dentin thirds and that the dentinal middle third was devoid of these structures. In an extensive retrospective review of the literature, we found that since 1962 Johansen and Park (17) described the presence of thin, sheetlike membranes at all the dentinal levels, except predentin. Afterward, several scientists studied dentin at the scanning electron microscopic level and observed similar structures (6–8, 16, 18–20). Microstructures described as bifurcation lateral branches and ramifications of the OP were observed to be associated with holes and fenestrations in the wall of the DT. In other studies, these structures were described close to the dentinoenamel junction and described as bifurcations of the DTs. Interestingly, results from other studies reported on a more or less complex microfibrillar network attached to the OPs within the DTs (6–8, 10, 13–16, 19–21). The aim of this work was to study and describe a microfibrillar and sheetlike network connecting DTs and OPs observed at different levels within the DTs of human teeth.
Materials and Methods We analyzed 30 caries-free human bicuspids from patients undergoing orthodontic treatment at the Orthodontics Clinic of the Postgraduate Studies Division in the Facultad de Odontologıa of the Universidad Nacional Autonoma de Mexico, Mexico City,
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Basic Research—Technology Mexico. All patients 18 years old or older and parents of the patients 17 years old or younger signed a letter of consent, donating their teeth to our institution for research purposes only. The ethics committee of our institution previously approved the protocol. Upper or lower bicuspids were extracted with regional anesthesia and minimal trauma using Xylocaine with 2% epinephrine (Zeyco, Guadalajara, Jalisco, Mexico). Immediately, we separated the crowns of the extracted teeth from the roots, making a groove at the cementoenamel junction with a watercooled, tungsten carbide bur and a high-speed handpiece. The final separation was made using a chisel and a hammer. Crowns were grooved in the mesiodistal direction, split in 2 halves, and immediately immersed overnight in Karnovsky fixative solution at 4 C. Then, they were rinsed in cacodylate buffer (pH = 7.4) and demineralized in 5% nitric acid aqueous solution. After critical point drying, crowns were mounted on aluminum stubs with colloidal silver, coated with a 20-nm-thick gold layer, and examined with a JEOL 2000 SEM (JEOL, Tokyo, Japan).
Results Patients were 15–21 years old with a mean age of 17.7 years (1.82 years standard deviation [SD]). The features of the dentinal tissue close to the pulp showed no differences with those previously reported. In the inner nonmineralized dentin, DTs contained OPs associated with numerous, thin, microfibrillar structures forming a dense network (Fig. 1A). In the inner dentinal third, 1 end of the microfibrils was attached to the OP, and the other extremity was connected to the DT wall. Spaces among these microfibers were small, and they were so numerous that they occupied a large portion of the DT, forming a dense net (Fig. 1B). In other areas, these fibers seemed to coalesce, becoming wider and forming thick structures and frequently looking like homogeneous sheetlike material (Fig. 1C). In the previously
mentioned areas, OPs were always in close contact with this microfibrillar network and were restricted within the periodontoblastic space. The most common direction of these microfibrils was from the surface of the OP to the dentinal wall, and in other instances, these microfibrils attached to both opposite internal surfaces of the DT. We were not able to see limits among the fibrillar or sheetlike structures and peritubular dentin. We observed that the base of the microfibril attached to the dentinal wall seemed to form a continuous structure among the dentinal tissue and the surface of the OP. In the different analyzed dentinal areas, the length, number, and diameter of the microfibrillar material varied widely. In the predentin area, microfibrils measured between 0.01- and 2.5-mm long with a mean of 1.09 mm (0.63 mm SD). The microfibril diameter was between 0.03 and 0.5 mm with a mean of 0.33 mm (0.33 mm SD); their number varied among 30 to 52 microfibers/10 mm2 with a mean of 42 microfibers/10 mm2 (6.8 microfibers/10 mm2 SD). Microfibers located in the inner third of the dentin-analyzed areas measured among 0.04- to 3.6-mm long (mean = 2.35 mm, 0.99 mm SD). Their diameter varied from 0.03–0.46 mm (mean = 0.32 mm, 0.13mm SD), and their number varied among 34–40 microfibers per 10 mm2 (mean = 36.6 microfibers per 10 mm2, 2.31 microfibers per 10 mm2 SD). In contrast, longitude of the microfibers located in the middle third was between 0.01 and 0.2 mm (mean = 0.07 mm, 0.05 mm SD); the microfiber diameter was among 0.03 and 0.52 mm with a mean of 0.05 mm (0.13 mm SD), and their number varied among 8 to 18 microfibers per 10 mm2 (mean = 11.9 microfibers/10 mm2; 2.9 microfibers/10 mm2 SD). The scarce microfibers located in the external dentinal third were almost all broken, and we propose this measurement will give false data; the diameter was between 0.01 and 0.02 with a mean diameter of 0.011 mm (0.007 mm SD). In addition, their number varied between 0 and
Figure 1. Nonmineralized dentin. (A) Multiple thin microfibrils of different diameters filling the dentinal tubules (arrows) (SEM, 10,000. Scale bar = 1 mm). (B) The inner third of the dentin. DT showing the presence of microfibrillar structures connecting the odontoblastic process to dentinal walls (arrows). (SEM, 2000. Scale bar = 10 mm). (C) DTs showing the presence of netlike structures formed by sheetlike structures (wide arrows) and some microfibrils (thin arrows). A hole is also seen (small arrow). (SEM, 3500. Scale bar = 5 mm). (D) The middle third of the dentin. We can see a lesser quantity of microfibrils in this zone (wide arrows), and some areas of the dentinal tubules are empty (thin arrows) (SEM, 2000. Scale bar = 10 mm). JOE — Volume 41, Number 9, September 2015
SEM of Dentin Fibrillar Structures
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Figure 2. Outer third of the dentin. (A) The dentinal tubules contain few microfibrils with an odontoblastic process (arrows) (SEM, 2000. Scale bar = 10 mm). (B) This photomicrograph shows several odontoblastic processes with no microfibrils (arrows) (SEM, 1500. Scale bar = 10 mm).
10 microfibers/10 mm2 (mean = 3.6 microfibers/10 mm2, 3.02 microfibers/10 mm2 SD). In the predentin area, very fine and numerous microfibrils formed a dense network. Comparatively, this framework was less dense in the inner dentinal third because the empty spaces of the net appeared wider and they were larger as we analyzed the more superficial dentin areas. In addition, in the middle third of the examined dentinal tissues, we were able to observe that compared with the inner dentinal third, a small number of microfibrils and sheetlike structures subsisted because we detected many areas devoid of the previously described microstructures (Fig. 1D). These microfibrillar and sheetlike structures were very scarce in the OPs located in the outer third of the dentin (Fig. 2A), and they almost disappeared as we observed DTs close to the dentinoenamel junction. In the more external dentinal areas, almost all DTs exhibited no microfibrils attached to their surface (Fig. 2B), or they were very few in number. Moreover, DTs in these areas rarely contained OPs. Interestingly, we were able to observe that in some areas of the middle dentinal third, the tip end of some OPs were smooth with a flat or round end. The corps of the terminal portions of these OPs showed few microfibrils attached on their surfaces (Fig. 3A) and in few instances, microfibrils were observed attached to the tip of the OPs (Fig. 3B). Additionally, we observed the presence of some round or nodular, smooth-surface structures of variable size. These structures were very common within the intertubular dentin and were observed as isolated or coalescent nodules. Sometimes they appeared as more or less enlarged structures and rarely as small nodules protruding to the tubular lumen. Frequently, the inner surface of the dentinal tubules appeared to be smooth or containing very fine microgranules. This feature was not in the outer third because granules in these areas were coarse.
Discussion For some time, dentin tissue has been extensively analyzed by means of scanning electron microscopy, and in the published studies
details of new dentinal structures were described (6–23). In 1964, Scott (24) described that OPs in the inner third of the dentin were associated with thin fibrils and thought they were OP ramifications. Reviewing previously published reports dealing with scanning electron microscopic studies on dentin architecture, we found that some of them (7, 8, 11, 13–16, 18–20) included photomicrographs showing microfibrillar structures of analogous morphology to the structures described and detailed in this study. In most of the reviewed reports, there were no comments on the presence or possible function of these structures, and the authors did not consider they were an important discovery. In some reports, their existence was commented on in few words (8, 13, 16, 18). Interestingly, only a few authors provided opinions on these microstructures, and they considered them as ramifications or lateral branches of the OP (14), a shredded fibrous network of the lamina limitans (7), branching of the lamina limitans (19), an anastomosing branching system (11), extensions of the lamina limitans (20), and collagen fibrils (15). Comparing features of the microfibrillar structures found in this study with the photomicrographic material in the previously published articles (7, 8, 11, 13–16, 18–20), we concluded they are the same. Our results showed these microstructures are microfibrils forming part of a more or less dense net; frequently, they were fine fibrils observed isolated, and, sometimes, these fibrils were wider, appearing as a thick membrane covering part of the inner surface of the DT and the OP. Sometimes, these structures were scarce, and isolated microfibrils in dentin areas were lacking OPs. Our results do not support the suggestion of Szabo et al (14) that microfibrils are branches or ramifications of the OP because branching and ramifications of the OPs are always related to holes in the dentinal wall connecting 1 OP within a DT with a neighbor OP (8, 11, 13, 20). In our reviewed material, this microfibrillar network was not in relation to holes in the surface of the analyzed DTs. For many years, it was considered that OPs within DTs are immersed in a liquid environment (the dentinal fluid), and according
Figure 3. Terminal portions of the odontoblastic process. (A) A photomicrograph showing some microfibrils (wide arrows) retained in the terminal portion of this odontoblastic process. Note that the end of 1 odontoblastic process is round (thin arrow) and the end of other 2 is not (small arrow) (SEM, 5000. Scale bar = 5 mm). (B) This photomicrograph shows the presence of some microfibrils attached to the terminal portion of the odontoblastic process (star) (SEM, 5000. Scale bar = 5 mm).
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Basic Research—Technology to this idea, OPs are ‘‘floating free’’ within this fluid. Our results show that the described microfibrillar network is part of a complex system, and we can suggest its function is to support the OPs and this microfibrillar system to maintain OPs in place within DTs. In this study, these microfibrillar structures never were associated with or seen within the lateral holes frequently described in the wall of the dentinal tubules. This finding strongly suggests they are not ramifications of the OP. Additionally, their fibrillar pattern also suggests that they are not related to the previously described lamina limitans. Under our experimental conditions, we were not able to discard a probable collagenous nature of the microfibrillar system described in this article. In addition, these microfibrils appeared attached to the surface of the OP; this feature suggests that the microfibrillar system forms part of the OP. Additionally, our findings strongly suggest that the netlike and sheetlike structures are also part of this system, helping the microfibrils in their function supporting and maintaining OPs secure to DT walls. The location of the previously mentioned microfibrillar netlike and sheetlike structures exclusively within DTs suggests that these microstructures could arise as a cellular product derived and secreted from the OP. It can be speculated that the findings we present can be associated with the method used, and they can be artifacts only. In this fashion, there are few studies on the changes produced by immersion of the dentin specimens in different acids for demineralization (25– 30). Demineralization by acids removes the dentin mineral material, and its first effect is to increase the diameter of the DTs removing the peritubular dentin (25) without a change in the center-to-center tubular distance (27). In 1993, Marshall et al (27) calculated that during dentinal demineralization, the peritubular depth changes were linear and moved at approximately 0.005 mm/s. Also, Paciornick et al (31) showed that the final tubule dentinal diameter is between 3.5 and 5.5 mm. It is also known that shrinkage is a constant phenomenon occurring during this process (25, 27,) and some studies showed that the loss of dentinal volume is different with the use of different demineralizing solutions (25, 26) and some substances as HEMA (hydroxyethylmethacrylate) and ethylene glycol can prevent this shrinking (25). It is important to comment that, according to the Habelitz et al (30) and Carvalho et al studies (26), this shrinkage and loss of volume are related to the removal of minerals and dehydration with the formation of more or less wider gaps between the collagen fibers. In this study, we used Karnovsky fixative and freeze-drying techniques; with the use of these techniques, some loss of volume is expected to be observed in our photomicrographs, and, as predicted, widening of the DTs and shrinkage of the structures are apparent. In our opinion, changes of the analyzed material are those expected, and we do not consider these changes severe because no distortion is observed in the micrographic material. There are some issues not completely understood that scanning electron microscopic studies can clarify. One of them is the so-called ‘‘butterfly effect.’’ Since 1983, butterfly images observed in root sections are considered an ‘‘optical phenomenon’’ associated with dentin sclerosis (32). The study of Russell et al (33) clarified this issue by concluding that under scanning electron microscopy, the buccolingual radicular area contains a higher number of dentinal tubules and that, clinically, restorations on buccal or lingual surfaces may achieve better retention and longevity than those on proximal surfaces. Moreover, the study by Ni~no-Barrera and Garzon-Alvarado (34) using scanning electron microscopy and a mathematical model suggested that spatial distribution of BMP-2 and Noggin determines the odontoblastic differentiation and dentinal tubule formation. Results from both studies explain the development of a higher or lower number of dentinal tubules in different radicular areas; this situation may be associated with lateral canal formation, which during endodontic treatment may influence an JOE — Volume 41, Number 9, September 2015
apparition of recurrent infections; in the Vieira et al (35) report, this structure was the cause of endodontic treatment failure. We suggest that new studies are necessary to confirm our results. To date, we are analyzing new material to search for new findings on human dentin architecture.
Conclusion We observed many microstructures as fine and thick microfibrils connecting the wall of the DT and the corps of the odontoblastic process, and in other instances, we observed netlike structures or sheetlike membranes in the nonmineralized predentin and the inner and middle thirds of the dentin tissue. They were found in higher numbers in the inner third of the dentin, and they were less numerous because the analyzed level was more superficial. According to our results, these microstructures may represent an unrecognized support system, which holds and secures the odontoblastic process within the dentinal tubule and is an integral part of the OP.
Acknowledgments The authors deny any conflicts of interest related to this study.
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