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RESEARCH REPORT |
1 Laboratorio de Biología Celular y Molecular, División de Estudios de Posgrado e Investigación, Facultad de Odontología, UNAM, Cd. Universitaria, 04510, México DF, Mexico;
2 Departamento de Biología, Facultad de Ciencias, UNAM, Mexico;
3 Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM; and
4 The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Israel;
* corresponding author, harzate{at}servidor.unam.mx
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: cementoblasts • cementum protein • periodontium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, a new cell line with cementoblastic characteristics was established from a human cementoblastoma tumor (Arzate et al., 1998). This cell line has been shown to express CAP and to form mineralized tissue in vitro similar to human cementum (Arzate et al., 1998, 2000). These findings indicated that the cementoblastoma cell line produces a 56-kDa protein species, that this species is CAP or a related molecule, and that it can act as an antigen for producing new antibodies capable of recognizing cementoblastic populations in vivo and in vitro. The purpose of this investigation was to test this hypothesis and to use the new antibody as a cementum biological marker.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Antibody Preparation
Partially purified CP preparation containing CP migrating with 56,000 Mr was obtained by electro-elution, as described previously (Arzate et al., 1996). New Zealand rabbits were immunized as described by Dunbar and Schwoebel (1990). Antibody production was monitored through ELISA and immunoblot. IgG antibodies were purified through protein A-sepharose chromatography (Sigma Chemical Co., St. Louis, MO, USA). The antibody fraction will be referred to as anti-CP antibody.
Cell Culture
Human alveolar bone and cementoblastoma-derived cells were obtained as previously described (Arzate et al., 1998). Human periodontal ligament and gingival fibroblasts were obtained from a premolar extracted for orthodontic reasons from a 25-year-old male patient. Cells were cultured by the conventional explant technique (Narayanan and Page, 1976). Cells were grown in DMEM medium supplemented with 10% FBS. All cell types from the 2nd passage were used for the experimental procedures.
Immunoaffinity Chromatography and Immunoblotting of CP
A 500-mL quantity of conditioned medium was collected from plates of each cell type containing cells at confluent density, and CP was immunopurified as described previously (Arzate et al., 1992b). Protein concentrations were determined by means of the Nano Orange Protein Assay Kit (Molecular Probes, Eugene, OR, USA). Cross-reactivity and expression of CP in human cementoblastoma, periodontal ligament cells, and alveolar bone-derived cells were assessed by immunoblotting as previously described (Arzate et al., 1992b), except that anti-CP antibody was used at a 1:300 dilution. For assessment of the specificity of anti-CP antibody, calfskin type I collagen (Boehringer Mannheim, Germany), bovine FN (Life Technologies, Rockville, MD, USA), and human bone extract were blotted and tested with anti-CP antibody. Antibodies against bovine type I collagen, FN, OPN, and BSP served as controls. To determine the uniqueness of the CP, we performed immunoblots with polyclonal antibodies against rat type I collagen (Chemicon International Inc., Temecula, CA, USA), human OPN (LF-123), human BSP (LF-100), both a gift from Dr. Larry W. Fisher (NIH, Bethesda, MD, USA), and human FN (Dako, Glostrup, Denmark). Immunoblots with anti-CP antibody were compared with those with a monoclonal antibody against bovine CAP (3G9), a gift from Dr. A.S. Narayanan (Seattle WA, USA).
Cell Attachment Assay
Periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts were plated at 2 x 104 density on 24-multiwell Costar plates not treated for tissue culture (Costar Corp., Cambridge, MA, USA) and coated with 1.0, 0.5, 0.2, and 0.1 µg/mL of immunoaffinity-purified CP. Cell attachment was evaluated according to Hayman et al.(1982). Wells coated with 5 µg/mL of calfskin type I collagen served as a positive control, and serum-free medium was the negative control.
Processing and Immunostaining of Human Tissues
Specimens were fixed in 10% formaldehyde. Hard tissues were decalcified with 10% EDTA, pH 7.4, dissolved in 0.5% formaldehyde at 4°C for 5 wks, and processed as previously described (Arzate et al., 1998).
Longitudinal and transverse sections 5 µm thick were cut and mounted in 2% 3-aminopropyltriethoxysilane-coated glass slides (Sigma Chemical Co., St. Louis, MO, USA). Sections were de-waxed in xylene and, before complete rehydration, were incubated with antigen retrieval solution as described by Shi et al.(1992). Immunocytochemical procedures were as described elsewhere (Arzate et al., 1998).
Immunostaining of Human Cementoblastoma, Periodontal Ligament, and Alveolar Bone-derived cells in vitro
Human cementoblastoma, periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts were plated at low density (5 x 102) on Lab-Tek chamber slides (Life Technologies, Rockville, MD, USA) allowed to attach overnight, and cultured for 3 days. Rabbit pre-immune serum or slides lacking first antibody were used as negative controls. Experiments were done in triplicate. We determined the number of cells cross-reacting with anti-CP antibody by scoring 5 different microscopic fields with a 20X lens.
Statistical Analysis
Cell attachment was expressed as % relative to positive control (type I collagen). Experiments were run in triplicate. We used one-way ANOVA to test variability and performed Tukey’s test to assess statistical significance at a level of P < 0.05 (n = 3). Experimental data for the immunostaining of human cementoblastoma, periodontal ligament, and alveolar bone-derived cells in vitro are presented as mean (n = 5) ± SE of 3 independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, the attachment of periodontal ligament cells, alveolar bone-derived cells, and gingival fibroblasts to plates incubated with immunopurified CP was dose-dependent. The number of periodontal ligament cells attaching to 0.1, 0.2, 0.5, and 1.0 µg/mL of CP represented 10, 26, 63, and 86%, respectively, relative to the positive control cultures, alveolar bone cells represented 8, 21, 53, and 89%, and gingival fibroblasts 8, 16, 30, and 42%. No statistical differences were observed between periodontal ligament cells and alveolar bone-derived cells. However, gingival fibroblast attachment was significantly lower at 0.2, 0.5, and 1.0 µg/mL of CP when compared with periodontal ligament cells and alveolar bone-derived cells (P < 0.05).
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). Anti-CP antibody cross-reacted with a single band of 56 kDa in protein(s) obtained from immunopurified conditioned medium of cementoblastoma cells (lane 1). Immunopurified protein(s) obtained from periodontal ligament cell conditioned medium immunoreacted positively (lane 2). Alveolar bone-derived cells immunopurified conditioned medium (lane 3) and human serum (lane 4) were negative. There was no cross-reactivity of the anti-CP antibody with type I collagen, FN, OPN, and BSP (data not shown). Immunoblots with immunopurified CP did not cross-react with antibodies against type I collagen, OPN, BSP, and FN (not shown). The monoclonal antibody against bovine CAP recognized a 70-kDa species in CP preparation (lane 5).
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). A few spindle-shaped periodontal ligament cells located close to a blood vessel in the region of cellular cementum as well as cementoblasts lining the cementum surface were positive (Fig. 3C). Cell clusters located between blood vessels and 1-3 cell layers from the cementoblast layer stained more intensely than the neighboring periodontal ligament cells (Figs. 3D, 3E). Elongated cells located within the endosteal spaces of the alveolar bone exhibited strong positive staining (Fig. 3F). Cementocytes embedded in the cellular cementum mineralized matrix and those close to the cementum-dentin junction were positive (Fig. 3G). Cementocytes in their lacunae and their interconnecting cytoplasmic processes showed strong immunostaining (Fig. 3H). Cells representing a transitory stage between cementoblasts and cementocytes just becoming embedded in the calcified cementum matrix were observed in cellular cementum. These cells stained more intensely than cells present in the periodontal ligament (Fig. 3I). Acellular cementum (cementoid layer) was intensely stained (Fig. 3J). Control sections where pre-immune rabbit serum was used were negative (Fig. 3K). Other human tissues, such as brain, liver, large intestine, pancreas, kidney, spleen, aorta, tendon, femur, rib, and osteosarcoma, immunoscreened for the expression of CP, revealed no significant immunofluorescence (not shown).
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). Positive cells represented approximately 95% (94.7 ± 2.6). In periodontal ligament cell cultures, 5.8 ± 0.9% cells stained with the anti-CP antibody (Fig. 4B). Cultures of alveolar bone-derived cells were largely negative, and they contained 3.4 ± 0.6% positive cells (Fig. 4C). Gingival fibroblasts were negative (not shown). Negative controls where non-immune sera were used as a first antibody were negative (Figs. 4D, 4E, 4F for cementoblastoma, periodontal ligament, and alveolar bone-derived cells, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-CAP monoclonal antibodies have been shown to recognize the cementoid phase of human cementum as well as a few cells located within the endosteal spaces of human alveolar bone (Arzate et al., 1992a). The present study demonstrated that CP is distributed throughout the entire root surface, including cellular and acellular cementum. The CP antibody positively stained cells located near the blood vessels in the periodontal ligament. In certain areas, clumps of positively stained cells which were localized between the blood vessels and active cementoid formation were observed. These clumps may represent expanding clones of the cementoblastic lineage. This assumption is further supported by a series of previous reports. First, the work of McCulloch and Melcher (1983) indicates that the progenitor pool in the periodontal ligament is located in the paravascular zone, whence cells migrate toward their target tissues, cementum, alveolar bone, and periodontal ligament. Second, the works of Liu et al. (1997) and Bar-Kana et al. (2000) demonstrated that there was a direct correlation between the capacity of periodontal ligament-derived progenitor clones to bind CAP and express CAP in culture and their capacity to produce mineralized cementum-like tissue in vitro. Analysis of these collective data suggests that the cementoblastic lineage expresses CP during its growth and maturation, both in vitro and in vivo, and that CP might be a key factor in these processes. This concept is supported by the findings of McCulloch (1985), McCulloch et al.(1987), Melcher et al. (1987), and Lang et al. (1995), who demonstrated that cells from the endosteal spaces of the alveolar bone migrate through vascular channels into the periodontal ligament and contribute to the paravascular pool. They also showed that alveolar bone-derived cells are capable of forming cementum-like tissue in vitro and in vivo, suggesting that at least some of the early progenitors of the cementoblastic lineage originate in the endosteal spaces of the alveolar bone.
In the present study, we found that 3% of alveolar bone cells stained positively for CP in vitro, and that some cells lining the endosteal spaces of the alveolar bone and the vascular channels also stained positively for CP. If it is assumed that CP is a marker for the cementoblastic lineage, then the results of our study strengthen the hypothesis that progenitors originating in the alveolar bone contribute to the cementoblastic lineage.
In the cementoid phase, cementoblasts just becoming embedded in the cementum matrix and cementocytes stained more intensely than the cementoblasts lining the cementum, indicating that cementum matrix formation and maturation are associated with CP synthesis and secretion. Since the calcification of cementum has been postulated to be under the control of cementoblasts lining the cementum and freshly embedded cementocytes, it is possible that CP plays an important role in the mineralization process of cementum.
Western blots showed that conditioned medium from cementoblastoma cells and periodontal ligament cells contained an antigen that cross-reacted with anti-CP antibody. However, this antigen was not detected in alveolar bone cell medium. This may be due to the small amount of protein loaded on gels. The poor yield from immunoaffinity chromatography may reflect low levels of CP production in these cultures, and that only 3% of the cells produce CP (Fig. 4C
). Nevertheless, these results indicate that osteoblasts and osteocytes do not express CP protein, in vivo and in vitro, and that cementoblasts and osteoblasts are therefore phenotypically different. This statement is supported by recent findings which revealed that the mineralized matrix deposited by putative cementoblasts is morphologically, compositionally, and ultrastructurally different from that deposited by human alveolar bone-derived cells in vitro and human bone marrow stromal cells (Arzate et al., 1998, 2000; Grzesik et al., 2000). Analysis of these data, together with our previous work on this cell line, demonstrates that cell populations with cementoblastic phenotype have the capacity to produce a protein (CP) that is immunologically related to CAP, since a monoclonal antibody against CAP cross-reacted with CP as a 70-kDa species. However, at this point it is not clear whether the difference in molecular size between these two species is due to differences in post-translational processing of the protein or whether the 70-kDa species is the precursor of CP. Since other molecules such as BSP have multiple molecular forms (Mintz et al., 1993) and CAP has also been reported as a 65-kDa species in the developing tooth germ (Saito et al., 2001), the possibility that CP and CAP could be related molecules appears valid. In summary, these studies demonstrate that CP is widely distributed throughout cementum, has the capacity to identify putative cementoblastic populations both in vivo and in vitro, and is immunologically related to CAP. Our studies also indicate that antibodies to CP could be useful to identify these populations and to elucidate the cellular and molecular mechanisms that control cementogenesis during homeostasis and wound healing.
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ACKNOWLEDGMENTS |
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Received August 14, 2001; Last revision June 11, 2002; Accepted June 17, 2002
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REFERENCES |
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| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
, alveolar bone-derived cells •, and gingival fibroblasts 
. Asterisk (*) indicates significant differences in cell attachment between periodontal ligament and alveolar bone-derived cells vs. gingival fibroblasts at P < 0.05.
