J Periodont Res 2003; 38; 502507 Printed in the UK.

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Immunohistochemical localization of bromodulin in the periodontium during cementogenesis and root formation in the rat molar
Matias MA, Li H, Young WG, Bartold PM. Immunohistochemical localization of bromodulin in the periodontium during cementogenesis and root formation in the rat molar. J Periodont Res 2003; 38; 502507. Blackwell Munksgaard, 2003 Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, bromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of bromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to bromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament bers insert into the alveolar bone and where the Sharpeys bers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that bromodulin is a signicant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hardsoft tissue interface during cementogenesis.

M. A. Matias1, H. Li1, W. G. Young1, P. M. Bartold2

1 University of Queensland, Department of Dentistry, Brisbane and 2University of Adelaide, CACDRC, Adelaide, Australia

P. M. Bartold, University of Adelaide, Colgate Australian Clinical Dental Research Center, Dental School, Frome Road, Adelaide South Australia 5006, Australia Tel: + 61 88303 3435 Fax: + 61 88303 6429 e-mail: mark.bartold@adelaide.edu.au Key words: fibromodulin; proteoglycan; cementum; periodontal ligament Accepted for publication March 3, 2003

Although considerable advances have been made in the last decade concerning the macromolecular composition of the extracellular matrix of cementum, many components have yet to be fully characterized (1). Recently, a

small leucine-rich proteoglycan, bromodulin was isolated from bovine periodontal ligament (2) and localized in bovine cementum (3). Fibromodulin has also been demonstrated in the periodontal ligament and cementum of

healthy and periodontally involved human teeth (4, 5). Fibromodulin is an acidic, leucine rich, 59-kDa proteoglycan that binds to collagen (6, 7) and inhibits collagen bril formation in vitro (6, 8).

Fibromodulin and cementogenesis It is thought to inuence brillogenesis by regulating collagen bril spacing and thickness (9). Fibromodulin is produced predominantly by chondrocytes and broblasts, and is detectable in many connective tissues. The core protein of bromodulin consists of repetitive leucine-rich amino acid sequences similar to biglycan and decorin. Fibromodulin, along with decorin, has a central role in the maintenance of the structure of collagen brils. In vivo experiments strongly suggest that small proteoglycans, such as bromodulin, are involved in adhesion, division, dierentiation and migration of cells and have functions related to the regulation of collagen organization of the pericellular and extracellular matrix. Thus, we hypothesized that bromodulin may be one of the key proteoglycans synthesized during cementogenesis. Therefore, the aim of this study was to examine, immunohistochemically, the distribution and localization of bromodulin during cementogenesis and root formation in a rat model. Because of the association between bromodulin and collagen, the distribution of bromodulin in the developing rat periodontium was compared to that of collagen type I. topes of the keratan sulfate-rich small leucine-rich proteoglycan, bromodulin (10). Anti-collagen Type I antibodies (LF-67) were generously provided by Dr L. W. Fisher, NIDCR, Bethesda, USA (11).
Immunohistochemical staining

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Sections were deparanized in xylene and rehydrated in 100%, 90% and 70% alcohol, distilled water and in phosphate-buered saline, consecutively. For the sections stained for bromodulin, an enzymatic pre-treatment with 1.25 U/ml chondroitinase ABC (Sigma, St Louis, MO, USA) in 0.01% bovine serum albumin, pH 7.2 for 10 min at 37C was performed to increase antibody accessibility to the epitopes (10). All primary antibodies were diluted in phosphate-buered saline containing 0.1% bovine serum albumin at concentrations previously determined

to give optimal staining (bromodulin 1 : 1000 and collagen type I 1 : 5000). Sections were washed in distilled water, and soaked in phosphate-buffered saline with 3% hydrogen peroxide (H2O2) for 15 min to block endogenous peroxidase activity. The sections were then blocked with 10% normal swine serum for 30 min at room temperature, and followed by incubation of the primary antibody overnight at 4C. After two washes in 0.1% Triton X-100/phosphate-buffered saline and once in phosphatebuered saline, the sections were incubated with a biotinylated secondary antibody followed by incubation with conjugated Streptavidin-peroxidase (Dako, Carpinteria, CA, USA) for 15 min. Specic immunostaining was visualized by incubation with 3,3-diaminobenzidine tetrachloride containing 0.015% H2O2 for 3 min and washed twice with distilled water. The sections were counterstained with

Table 1. Summary of the formation of the periodontal tissue compartments of the rat maxillary rst molar at 3, 5 and 8 weeks Cementum/dentine 3 weeks Formation of acellular cementum along the root dentine. Cellular cementum formation is initiated at the apical portion of the developing root. 5 weeks Complete formation of acellular cementum in the coronal half of the root with increased thickness. Almost complete formation of root dentine. Cementocytes in the forming cellular cementum present at the apical half of the root with increased thickness of cellular cementum. Completion of root dentine and mature cementum.

Material and methods

Animals
8 weeks

In this study, three normal Lewis rats for each age group (3, 5 and 8 weeks old) were used, following approval from the Animal Ethics Committee, University of Queensland. The maxillary molars, together with the surrounding alveolar bone and gingival tissues were block dissected and xed. Serial 5 lm longitudinal sections were then cut in the bucco-lingual plane of the tooth.
Antibodies

Periodontal ligament 3 weeks Collagen bers are forming and attaching to nearby newly formed bone and attaching into acellular cementum along the root dentine. Oblique orientation of cells and brous bundles. 5 weeks Increased density of PDL cells and PDL space. Increased cellular activity apical to the CEJ and along the alveolar crest region. PDL cells and collagen bers are well organized. PDL space width remains constant and principal collagen bers well organized.

8 weeks

Alveolar bone 3 weeks

Active bone formation stage. Large population of active osteoblasts at alveolar bone edges and inside bone marrow elements and spaces. Increase in mineralized bone with high activity of osteoblasts localized at the alveolar bone crest region. Decrease of bone marrow spaces and elements. Mature alveolar bone.

Anti-bromodulin antibody was a generous gift from Dr A. Plaas (Shriners Hospital for Crippled Children, Tampa, Florida, USA). This antibody recognizes core protein epi-

5 weeks

8 weeks

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Matias et al. Mayers hematoxylin solution for 1 min, rinsed in running tap water, dehydrated in ethanol (70%, 90%, 100%, respectively) for 3 min each and cleared with xylene. The slides were mounted in DePeX mounting medium (BDH Laboratory Supplies, Poole, England). All the sections were viewed and photographically recorded using an Olympus BX50 Microscope (Tokyo, Japan).
Controls

Non-immunized mouse serum and phosphate-buered saline were substituted for the primary antibody in negative control sections. Cartilage tissues located within the craniofacial complex were used as a positive control to check for the accuracy of staining of the antibromodulin antibody.
Assessment
Fig. 1. Distribution of bromodulin in the developing periodontal tissues of the maxillary molar teeth in a 3-week-old rat (A), 5-week-old rat (B) and 8-week-old rat (C). Immunoreactivity to bromodulin was evident in the periodontal ligament in all molar sections. The intensity of the staining increased with increasing tissue maturity. In 5-week-old rats (B), there was a marked intense positive stain in the extracellular matrix where the periodontal ligament bers insert into the alveolar bone and where the Sharpeys bers insert into the cementum (arrows). Original magnication 20; bar 100 lm. B, bone; D, dentin; AC, acellular cementum; PDL, periodontal ligament.

An assessment of the spatial distribution and localization of the antibody staining was made. The intensity of the staining was determined in a semiquantitative assessment. Immunoreactivity of the antibody in the major tissues and cells in the periodontium was given a score (0, +/,+ , ++ , +++). The score ranged from negative stain (0) to very strong positive stain (+++). A score of +/ represented tissues/cells which were weakly positive but some negative, a score of + represented tissue/cells which were positive with weak staining and a score of ++ represented tissues/cells which were positive with strong staining. As this was a descriptive study, statistical analysis of the data was not undertaken.

Results
By using rats aged 3, 5 and 8 weeks it was possible to determine the distribution of bromodulin and collagen type I at various stages of root formation and cementum deposition. A summary of the features of root and cementum formation at each of these stages is shown in Table 1. The expression of bromodulin in the developing rat molar periodontium produced a specic pattern. Fibro-

Fig. 2. Fibromodulin staining in cartilage in a 3-week-old rat (A), 5-week-old rat (B) and 8-week-old rat (C). The intensity of the staining decreased with increasing tissue maturation, and as the cartilaginous plate was replaced by bone tissue. Original magnication 40; bar 50 lm.

Fibromodulin and cementogenesis

Table 2. Immunoreactivity of bromodulin and collagen type-1 in the developing periodontium Fibromodulin Age (weeks) Tissue/cells Cementoblasts Cementocytes Cementoid Cellular cementum Acellular cementum Osteoblasts Osteocytes Osteoid Bundle bone Bone matrix Periodontal ligament cells Periodontal ligament matrix Gingival broblasts Gingival matrix 3 0 0 0 0 0 +/ 0 +/ 0 0 + ++ ++ ++ 5 8 Collagen type I Age (weeks) 3 5 8

505

+/ +/ 0 0 0 0 ++ ++ ++ 0 0 0 0 0 + +/ +/ +/ 0 0 +/ + 0 ++ 0 0 ++ 0 0 ++ ++ +++ + + +/+ + + + + + + ++ ++ + +++ +++ +

+/ +/ +/ +/ +++ +++ ++ ++ + + +/ +/ +/ 0 ++ +++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ +++

evident in the extracellular matrix of the mineralized acellular and cellular cementum (Fig. 3). When animals of dierent ages were compared it was noted that the general intensity of the bromodulin staining increased with increasing tissue maturation (Fig. 1). Interestingly, staining within the gingival tissues also showed some degree of site specicity. There was an intense band of bromodulin staining present in the palatal gingival connective tissue in all sections. In comparison, there was no evident staining for bromodulin in the buccal gingival connective tissue. A similar pattern of distribution was seen for collagen type I (Fig. 4AD).

0, negative stain in all rat sections; +/, some weakly positive/some negative; +, positive stain in all rat sections with weak staining; + +, strong stain and/or more cells positive; + + +, very strong stain and/or more cells positive.

Discussion
To date, the role that proteoglycans play during periodontal development has not been fully elucidated. This study has demonstrated that bromodulin, a small leucine-rich proteoglycan, can be localized in the periodontium of molar rat teeth during cementogenesis and tooth root development. By studying three dierent stages of root development (initiation, development and maturation), this study expands on previous studies which have considered the distribution of bromodulin only in mature cementum of adult and aged teeth (35). Fibromodulin was localized in the periodontal ligament, cementoid, pericellular areas surrounding cementoblasts and osteoblasts, osteoid and gingival connective tissue. In these areas, bromodulin appeared to be closely associated with the distribution of collagen type I. Several studies have shown that bromodulin binds to collagen type I and II (6, 7), whereby the core protein of bromodulin binds to collagen monomers, and the attached keratan sulfate chains appear to control bril diameter and interbrillar spacing inuencing brillogenesis (9). The intense staining of bromodulin at the hardsoft tissue interface of boneperiodontal ligament and cementumperiodontal ligament is in agreement with other studies in which adult periodontal tissues have shown

Table 3. Summary of immunoreactivity of bromodulin and collagen type I in other tissues/ cells associated with the developing periodontium Fibromodulin Age (weeks) Tissue/cells Chondroblasts Cartilage Blood vessels Pre-dentine Dentine Odontoblasts Dental pulp 3 ++ ++ 0 + 0 +/ +/ 5 +++ ++ 0 + 0 +/ +/ 8 ++ +/ 0 ++ 0 +/ +/ Collagen type I Age (weeks) 3 + + + ++ ++ + + 5 +/ +/ + +++ ++ + + 8 +/ 0 + +++ ++ + +

0, negative stain in all rat sections; +/, some weakly positive/some negative; +, positive stain in all rat sections with weak staining; + +, strong stain and/or more cells positive; + + +, very strong stain and/or more cells positive.

modulin was localized in several areas in the periodontium including the periodontal ligament and the gingival connective tissue (Fig. 1). In addition, there was some staining in the periosteum, osteoid, cementoid, pre-dentine, walls of endosteal spaces and blood vessel walls. Chondrocytes and newly formed cartilage also reacted positively to the bromodulin antibody. However, the intensity of staining in cartilage decreased as the cartilaginous plate was gradually replaced by bone tissue (Fig. 2). Tables 2 and 3 show summaries of the immunoreactivity of

the distribution of bromodulin and collagen type I in the developing periodontium and in other tissue compartments associated with root development. Within the periodontium, variations in staining intensity for bromodulin were noted. An intense positive stain was observed in the extracellular matrix where the periodontal ligament bers insert into the alveolar bone and where the Sharpeys bers insert into the cementum. Cementoblasts and osteoblasts showed mild staining for bromodulin. There was no staining

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Matias et al. Fibromodulin may also potentially inuence mineralization at these hard soft tissue interfaces; however, the evidence for this is equivocal. In vitro studies have shown that dentinal proteoglycans can inhibit mineralization (14), and maintenance of tubular pathways in odontoblastic process may be partly regulated by glycosaminoglycans (15). It has been suggested that bromodulin expression in bovine precementum indicates an inhibitory role in matrix mineralization (3). Notably, in the present study, bromodulin staining appeared to increase during development of cementoid and this would appear to corroborate its role in inhibiting mineralization in this early form of cementum. As noted in the present study, within the cementum mineralized matrix proper, there is no evidence of bromodulin (35). In conclusion, this study has demonstrated that bromodulin is expressed in the periodontal ligament during cementogenesis. Its particularly strong localization at the cementum/periodontal ligament interface implies an important role in the formation and maintenance of Sharpeys bers and, possibly, a role in regulating the mineralization process. Further investigations are required to determine the specic role of bromodulin during periodontal development.

Fig. 3. Distribution of bromodulin in the mineralized acellular (A) and cellular (B) cementum in a 5-week-old rat. There was no evident staining in the extracellular matrix of acellular (arrow in panel A) and cellular (arrow in B) cementum. Original magnication 40; bar 50 lm.

Acknowledgments
Fig. 4. Distribution of bromodulin (A and B) and collagen type I (C and D) in the gingival connective tissue in a 5-week-old rat. There was an intense band (arrows) of bromodulin staining in the palatal aspect (A) of the molar tooth compared with negligible staining in the buccal aspect (B). A similar pattern of distribution (arrows) was observed for collagen type I staining (C shows the palatal and D the buccal aspect). Original magnication 20; bar 100 lm. E, epithelium; GCT, gingival connective tissue; B, bone; D, dentin.

This study was supported by the Australian Dental Research Foundation (ADRF).

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Fibromodulin and cementogenesis

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