Calcif Tissue Int (2007) 81:39–45 DOI 10.1007/s00223-007-9027-z
Immunohistochemical Identification of Decorin and Biglycan in Human Dentin: A Correlative Field Emission Scanning Electron Microscopy/Transmission Electron Microscopy Study G. Orsini Æ A. Ruggeri Jr. Æ A. Mazzoni Æ V. Papa Æ G. Mazzotti Æ R. Di Lenarda Æ L. Breschi
Received: 20 December 2006 / Accepted: 26 February 2007 / Published online: 23 May 2007 Springer Science+Business Media, LLC 2007
Abstract Decorin and biglycan, two small leucine-rich proteoglycans, have been proposed to play important roles in matrix-mediated formation of mineralized tissues, and their three-dimensional arrangement in human dentin is still not completely understood. The aim of this study was to immunohistochemically analyze the distribution of decorin and biglycan in human predentin/dentin organic matrix under a high-resolution field emission in-lens scanning electron microscope (FEI-SEM) and a transmission electron microscope (TEM). Tooth dentin specimens were submitted to either a preembedding or a postembedding immunolabeling technique using primary antibodies antidecorin and antibiglycan and gold-conjugated secondary antibodies. Correlative FEI-SEM/TEM observations showed that the two antibodies yielded a similar labeling pattern over the processes of odontoblasts and the predentin. Decorin and biglycan were mainly associated with the collagen fibers within the predentin layer, revealing a moderate immunoreaction that was significantly higher compared to the one observed on dentin. Thus, a generally weak labeling for decorin was found in dentin, which, however, was significantly higher on odontoblast processes within dentinal tubules than in intertubular dentin. On the
G. Orsini Department of Biomorphology, University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy A. Ruggeri Jr. A. Mazzoni V. Papa G. Mazzotti Department of Anatomical Sciences, University of Bologna, Via Irnerio 48, 40100 Bologna, Italy R. Di Lenarda L. Breschi (&) Department of Biomedicine, University of Trieste, Via Stuparich, 1, 34129 Trieste, Italy e-mail:
[email protected]
other hand, biglycan immunolocalization on dentin revealed few gold particles rather uniformly distributed, without showing significant differences between tubular and intertubular regions. In conclusion, this study reveals distinct distribution patterns of decorin and biglycan and their relation with collagen. Decorin’s and biglycan’s precise roles within prematrix and mineralized matrix in human teeth should be further clarified. Keywords Decorin – Biglycan – Dentin Electron microscopy Immunohistochemistry
Introduction In addition to collagen, the dentin extracellular matrix (ECM) contains multiple noncollagenous proteins, proteoglycans (PGs), and lipids [1, 2]. Among these, PGs are deemed to play structural and metabolic functional roles in soft and calcified tissues, being key components of the mineralization process of dentin and bone [3–5]. Classically, PGs can be defined as one or more glycosaminoglycans (GAGs) covalently attached to specific core proteins [6]. The GAG side chains attached to the core proteins dictate the functional characteristics of PGs. Biochemical and immunohistochemical studies on predentin and dentin PGs indicated that predominantly PGs belong to the small leucine-rich PG (SLRP) family. Moreover, a large aggregating PG, called versican, has also been described in the form of degradation products in predentin [6] and more recently in dentin [7]. The distinctive feature of SLRPs is the presence of 7–24 leucine-rich tandem repeats in the core protein. Dentin SLRPs are mainly chondroitin sulfate (CS)-rich, with decorin (DCN) and biglycan (BGN) being the most prominent
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members, whereas the keratan sulfate-rich fibromodulin and lumican showed a more limited distribution [1, 6–9]. Much information gathered on the temporospatial distribution of SLRPs during dentinogenesis comes from immunohistochemistry [10], which effectively provides in situ identification of specific substrates by means of a wide range of monoclonal and polyclonal antibodies tagged with specific labels [11]. DCN and BGN can bind type I procollagen and collagen, thus regulating fibrillogenesis during dentinogenesis [10, 12, 13]. DCN usually carries one GAG chain, where CS predominates in hard tissues [14] and dermatan sulfate (DS) in soft tissues. BGN carries two GAG CS/DS chains [5], with CS being predominant in calcified tissue, as for DCN [6]. Despite their high level of homology at the amino acid level, DCN and BGN expression patterns are different [10, 15]. An immunohistochemical study on rat incisors showed that BGN is uniformly distributed throughout predentin and forming circumpulpal dentin, whereas DCN displays a sharp increase near distal predentin, where mineralization occurs [12]. Yoshiba et al. [16] reported negative DCN immunolocalization in human teeth in mantle dentin and initial predentin, whereas intense activity along the calcification front and dentinal tubules was present. All these reports may suggest that DCN can be more involved in dentin mineralization than BGN; however, direct and clear evidence of their precise roles remains to be obtained [7, 10, 12]. This study aimed to investigate labeling patterns of BGN and DCN within human predentin and dentin ECM using a high-resolution field emission in-lens scanning electron microscope (FEI-SEM) and a transmission electron microscope (TEM). For three-dimensional protein distribution within the dentin ECM, a preembedding immunohistochemical technique was applied to calcified unfixed dentin samples, which were then examined using FEI-SEM. For ultrastructural and protein compositional analyses, a postembedding colloidal gold immunocytochemical technique was applied to ethylenediaminetetraacetic acid (EDTA)-decalcified thin dentinal sections of human molars, which were then observed by TEM. The research hypothesis tested was that DCN and BGN are homogenously distributed within the predentin/dentin ECM.
Materials and Methods Ten human third molars scheduled for extraction for orthodontic reasons in subjects with a mean age of 18.9 years were processed after signed informed consent under a protocol approved by the Ethics Committee of the University of Bologna (Italy) was obtained. Roots were removed with a low-speed diamond saw, and each tooth was transversely sectioned to obtain 2-mm-thick dentin disks at
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G. Orsini et al.: Immunohistochemistry of Decorin and Biglycan
the medial level of the molar pulpal chamber. Tooth fragments were randomly assigned to the following groups: the FEI-SEM group, in which pulp was partially removed using a dental excavator spoon and disks were fractured to expose the inner surface of the pulpal chamber and processed for a preembedding immunohistochemical procedure, and the TEM group, in which specimens were immediately fixed, decalcified, and processed for postembedding immunocytochemistry. Preembedding Technique and FEI-SEM Tissue Processing Unfixed specimens of the FEI-SEM group were processed for a preembedding immunolabeling procedure in accordance with Breschi et al. [17], using two polyclonal primary antibodies, a human immunoglobulin G (IgG) antiDCN (LF-122) and a human IgG anti-BGN (LF-51) [14, 18–20]. In brief, specimens were immersed in 0.05 M Tris HCl-buffered solutions at pH 7.6, preincubated with normal goat serum, and incubated either with anti-DCN or with anti-BGN primary antibodies. Gold labeling was performed using secondary antibodies conjugated with 15 nm gold particles (goat IgG anti-rabbit IgG gold-conjugated; British BioCell International, Cardiff, UK) for DCN and BGN identification. Fixation was performed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2 for 4 hours; then, specimens were dehydrated in an ascending ethanol series and air-dried with hexamethyldisilazane. Specimens were mounted on microscope stubs and coated with a 1-nm-thick layer of evaporated carbon using a Balzers Med 010 Multicoating System (Bal-Tec, Balzers, Liechtenstein). Observations were performed under a JEOL (Tokyo, Japan) JSM 890 FEISEM at 7 KV accelerating voltage and 1 x 10–12 amp probe current. Final images were obtained with both back-scattered and secondary electron signals, and measurements were performed utilizing the proprietary image-analysis software of the microscope (JSMSCSI, JEOL, Milan, Italy). Controls consisted of dentin specimens processed as previously described but incubated (1) only with secondary antibodies, (2) after removal of the organic matrix with 5% sodium hypochlorite [21], (3) by omitting or substituting the primary antiserum with nonimmune serum, or (4) using unrelated primary antibodies. Tissue Processing for the TEM Group and Postembedding Technique Disks of the TEM group were immediately fixed in 0.4% paraformaldehyde-0.1% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2 and kept overnight at 4C, washed for 1 hour in the buffer alone, and decalcified using 4.13%
G. Orsini et al.: Immunohistochemistry of Decorin and Biglycan
EDTA for 3 months, followed by washing with 0.1 M cacodylate buffer at pH 7.2. Samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin (London Resin, Theale, UK). Semithin sections (1 lm) were cut with glass knives on a Reichert Jung (Heidelberg, Germany) Ultracut E ultramicrotome and stained with toluidine blue. Selected areas of the 1-lmthick sections were trimmed for ultrathin sectioning (80 nm) using a diamond knife and mounted on formvar carbon-coated nickel grids. Grid-mounted tissue thin sections were processed for immunocytochemical labeling following a postembedding technique in accordance with Breschi et al. [21]. Similar to the previously described preembedding procedure, immunolabeling was performed using the same primary antibody, anti-DCN or anti-BGN, and the same secondary antibody (goat IgG anti-rabbit IgG conjugated with 15 nm colloidal gold particles). Grids were stained with 4% uranyl acetate and lead citrate for examination in a JEOL (Tokyo, Japan) 1010 TEM operated at 60 kV. The TEM was connected to a MegaView III digital camera equipped with the Analysis Imaging System (Soft Imaging System GmbH, Munster, Germany). Controls consisted of sections incubated (1) with secondary antibodies only [22], (2) by substituting the primary antiserum with nonimmune serum, or (3) using unrelated primary antibodies. Quantitative Evaluation Gold particles were scored on a square area, 2 · 2 lm, on TEM micrographs (n = 40, magnification ·22,000) of different compartments (odontoblast processes, predentin,
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dentin-intertubular dentin, and dentinal tubules) and expressed as labeling index (LI), i.e., number of gold particles per square micrometer of visible organic network [12, 23]. Both DCN and BGN data were not normally distributed (Shapiro-Wilk statistic); thus, differences in DCN and BGN levels across groups were tested using the KruskalWallis test. The level of statistical significance was set at P £ 0.05.
Results Correlative FEI-SEM/TEM micrographs showed the morphological organization of human predentin and dentin ECM. SEM showed the three-dimensional organization of collagen fibrils in predentin (Fig. 1A) and the presence of dentinal tubules in dentin (Fig. 1B). By TEM it was possible to visualize aligned odontoblasts separated from dentin by a layer of predentin organic matrix (Fig. 1C). Mature dentin was characterized by packed dentinal tubules containing the cytoplasmic extensions of the odontoblasts; intertubular dentin was located between the dentinal tubules (Fig. 1D). FEI-SEM analysis allowed visualization of a moderately intense labeling for DCN over predentin (Fig. 2A) and a moderate to weak reaction over dentin (Fig. 2B). Immunoreaction for BGN was moderate in predentin (Fig. 2C) and weak in dentin (Fig. 2D). A clear spatial threedimensional relation was found between DCN or BGN and the collagen fibrils. Similar findings were obtained under TEM, with DCN and BGN yielding essentially the same labeling pattern
Fig. 1 Micrographs illustrating the morphology of human dentin specimens. SEM preparations of (A) predentin, showing its intricate network of collagen fibril bundles, and (B) dentin with a characteristic dentinal tubule (Dt). Dentin located between dentinal tubules is called ‘‘intertubular dentin’’ (iD). TEM images illustrating (C) aligned odontoblasts (Od) that extend their processes throughout the predentin layer and (D) dentin presenting numerous odontoblast processes (Odp) running in the dentinal tubules. N, nucleus
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Fig. 2 FEI-SEM of predentin and dentin after immunolabeling with polyclonal antibodies for decorin (DCN, in A and B) and biglycan (BGN, C and D). The images were obtained by a combination of secondary electron and back-scattered electron signals. (A) There was a moderately intense reaction for DCN (arrows) over the collagen fibrils forming predentin. (B) Gold nanoparticles (arrows) were also observed in intertubular dentin (iD); Dt, dentinal tubules. (C) Label for BGN was moderate and dispersed over predentin (arrows). (D) A few gold particles were detected within the dentin matrix, being mainly over the intertubular dentin
near the odontoblasts. The odontoblast processes showed a positive immunoreaction for both DCN and BGN (Fig. 3A, D), and gold particles on cytoplasm of the odontoblasts facing the dental pulp were seen (data not shown). Qualitative estimation of DCN immunolocalization showed a moderate labeling in predentin, where the collagen fibrils became more packed (Fig. 3B). BGN was uniformly distributed and detected over the bundles of collagen fibrils within the entire thickness of the predentin layer (Fig. 3E). The mineralized dentin region showed a generally weak immunoreaction for both antibodies, significantly lower compared to the one revealed in odontoblast processes and predentin. Tables 1 and 2 report the quantitative evaluation of the gold-antibody complexes for DCN and BGN, respectively, in the different regions evaluated (odontoblast processes, predentin, and dentin–intertubular dentin and dentinal tubules). While DCN labeling was significantly higher over the odontoblast processes inside the dentinal tubules (Table 1; Figs. 2B, 3C) compared to the intertubular dentin, the BGN labeling pattern was rather diffuse and more uniformly distributed, some gold particles being revealed inside the tubules and over the intertubular dentin (Table 2; Figs. 2D, 3F). Control specimens showed no labeling, thus confirming no cross-reaction between the secondary antibody and (1) the predentin/dentin substrate, (2) nonimmune serum, and (3) unrelated primary antibodies. FEI-SEM preparations of control specimens rarely showed scattered distributed gold particles over the dentin (Fig. 4A, B). Also, in controls of TEM preparations, only a few randomly distributed gold particles were seen over the tissue sections (Fig. 4B, D).
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Discussion A comprehensive understanding of the formation, structure, and properties of calcified tissues requires characterization of the distribution and interrelationship of ECM constituents [24]. Previous evidence of PG localization in dentin showed the distribution to be homogenous within predentin and moderate in dentin. Actual acquisitions have drawn a more complex situation, which yielded to a nonhomogenous distribution of PG populations in predentin/ dentin ECM, thus suggesting different and specific functions in each respective location [6]. Biochemically distinct pools of PGs were reported [7, 25]; i.e., a major CS PG (with relatively small molecular weight) was extracted from matrices of unmineralized predentin and mineralized dentin, whereas a second PG containing CS chains (but with a larger molecular size) was described as associated with predentin only [25]. Waddington et al. [7] identified different profiles of PGs associated with extracts of predentin, predentin/dentin interface, and dentin. Western blotting with bovine antiDCN and anti-BGN confirmed the presence of these two species of SLRPs within the three fractions [13]. However, the constituent GAG chains present within the predentin contained a higher proportion of DS compared to those identified in dentin matrix solely substituted with CS [7, 13]. It has been recently accepted that PGs synthesized within the predentin matrix play important roles in matrix formation and in prevention of premature mineralization; in fact, the large aggregating PG versican has been proposed to possibly act as a temporary scaffold in the capture
G. Orsini et al.: Immunohistochemistry of Decorin and Biglycan
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Fig. 3 TEM showing immunoreactivity for decorin (DCN in A–C) and biglycan (BGN in D–F) after a postembedding procedure. (A) DCN labeling was moderately intense and diffuse over the odontoblast process (Odp) in predentin, and few gold particles were present over the nuclei (N). (B) There was a positive immunoreaction for DCN in distal predentin, where the collagen fibrils (CF) became more packed (arrows) near the mineralization front. (C) Intertubular dentin (iD) showed weak labeling for DCN, whereas the odontoblast process inside the dentinal tubule showed a moderately strong immunoreaction (arrowheads). Immunolocalization of BGN was moderate over the odontoblast processes (D) and (E) diffuse and mainly detected over the collagen fibrils throughout the predentin (arrows); weak labeling was observed in the intertubular dentin, some gold particles being localized in the peritubular dentin (arrowheads)
Table 1 Number of gold particle-antibody complexes/lm2 (LI) in different regions of molar human teeth after labeling for decorin
Table 2 Number of gold particle-antibody complexes/lm2 (LI) in different regions of molar human teeth after labeling for biglycan
Specimen area
Decorin (mean ± SD)
Specimen area
Biglycan (mean ± SD)
Odontoblast processes
8.3 ± 1.7a
Odontoblast processes
6.1 ± 2.3a
Predentin
8.5 ± 1.9a
Predentin
6.0 ± 1.8a
Dentin (intertubular + tubules)
6.1 ± 3.4b
Dentin (intertubular + tubules)
1.8 ± 1.0b
Intertubular dentin Dentinal tubules
2.9 ± 0.8c 9.3 ± 1.6a
Intertubular dentin
2.3 ± 1.1b
Dentinal tubules
1.4 ± 0.6b
Different superscript letters indicate statistical difference (P < 0.05)
Different superscript letters indicate statistical difference (P < 0.05)
of space during the initial formation of ECM [7, 26–28]. On the other hand, the CS-substituted SLRPs within dentin may function in the regulation of the mineralization process [26]. The proposed roles for DCN and BGN include influence in collagen stabilization [29], collagen fibrillogenesis, calcium binding potential, interaction with hydroxyapatite, and inhibition of crystal growth [6, 12]. BGN was an effective inhibitor of mineral accretion in seeded growth experiments performed on bone SLRPs in a gelatin gel system, whereas DCN did not influence seeded growth [30]. Goldberg et al. [15] recently investigated
BGN and DCN deficiency on predentin of mouse molars, showing that BGN (but not DCN) modulates collagen fibrillogenesis during dentinogenesis and that DCN (and to a lesser extent BGN) promotes dentin mineralization [see also 19]. Correlative FEI-SEM and TEM immunohistochemical analysis allowed for the identification of DCN and BGN similar to that achieved by conventional histochemistry but with sharper indications of their specific localization. A previously reported preembedding FEI-SEM approach was used to characterize the spatial relationship of PGs and
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Fig. 4 Micrographs illustrating control specimens obtained using secondary antibodies only. SEM preparations of (A) a dentinal tubule (Dt), showing only two gold particles (arrowheads) and no immunoreaction over the intertubular dentin (iD). At high magnification (B), no labeling (arrowheads) of collagen fibers (CF) was detected. TEM images illustrating (C) no labeling of the collagen fibrils (arrowheads) within the predentin layer and (D) mature dentin revealing no crossreaction with the secondary antibody; a few randomly sparse gold particles were sometimes observed (arrowhead). Odp, odontoblast process
collagen in dentin [17, 21]. Then, to correlate FEI-SEM observations with TEM analyses, a postembedding technique was carried out in EDTA-demineralized dentin specimens [22]. This correlative approach was also preferred in the present investigation since EDTA demineralization has been shown to preserve PG structure, thus allowing easier thin sectioning of the samples and better preservation of the dentin in the embedding procedure, thus avoiding possible modifications due to the drying method [21]. Although previous works [9, 12, 13] have augmented the knowledge regarding the nature and precise distribution of SLRPs in dentin of rat incisors and bovine teeth, few studies have been directed at determining human dentin DCN and BGN localization. To date, only DCN immunolocalization has been investigated in human teeth [16]. Positive staining for DCN was demonstrated in odontoblast cell bodies and their processes in predentin, reaching an intensification along the predentin-dentin junction and showing no reaction in the predentinal matrix. Our findings are in partial agreement with this report, thus suggesting that DCN may be synthesized by odontoblasts, transported through their processes, and mainly secreted at the level of the predentin-dentin junction [31]. However, we also observed some fairly homogenous moderate labeling for DCN in the proximal predentin. This may be related to the fact that the tested primary polyclonal antibody may partially recognize not only the DCN mature form but also its proform, which has been reported to be diffuse in predentinal matrix and to increase toward the mineralization front [12]. This latter localization has a possible correlation between the presence of matrix metalloproteinases and deglycosylation of DCN, the protein core epitopes
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increasing in the distal part [6, 32]. In fact, a threefold increase of DCN labeling was detected between the proximal and distal regions of predentin by Septier et al. [12], while secreted BGN remained constant throughout predentin and the proform decreased by one-half in metadentin and dentin, BGN remaining uniform and DCN having a further rise in dentin. The mature dentin of our specimens generally showed a significant decrease of labeling compared to the one found in predentin. Indeed, there was homogenous weak labeling for BGN (sometimes detected in peritubular regions and intertubular dentin), whereas the reaction for DCN was not uniformly distributed, being more strongly associated with odontoblast processes inside dentinal tubules than in intertubular dentin. Since the results of the immunogold identification of DCN and BGN have led to a labeling pattern (on human dentin) similar to the one described by Septier et al. [12] (on rat incisors dentin), we suggest that also in human teeth these two SLRPs might have a role in dentin mineralization. To the best of our knowledge, this is the first TEM/FEISEM report on DCN and BGN labeling patterns in human dentin, and the research hypothesis of their homogenous distribution should be rejected since they show different expression in predentin/dentin ECM. In particular, our quantitative estimation demonstrates that DCN immunoreaction over the mineralized dentin mainly accounts for its localization inside the dentinal tubules (P < 0.05). Within the limits of our dentin specimens and the regions evaluated, these results may be of value in suggesting an important role of DCN in the mineralized matrix and in the predentin adjacent to the mineralization front, whereas BGN labeling distribution indicates its prevalent involve-
G. Orsini et al.: Immunohistochemistry of Decorin and Biglycan
ment during prematrix formation. Future studies will be carried out to distinguish the different forms of these SLRPs and to evaluate their modifications throughout the prematrix/matrix transition. Acknowledgements The authors thank Mr. Marcello Piccirilli for extensive technical assistance, Mr. Aurelio Valmori for photographic work, and Prof. Lamberto Manzoli for the statistical analysis. We thank Dr. Larry Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD) for generously donating the anti-DCN (LF122) and anti-BGN (LF-51) antibodies. The investigation was supported by grants (ex-60%) from Italian Ministry of Education, University and Research, Italy.
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