ORIGINAL Bussadori ARTICLE et al
Production of Extracellular Matrix Proteins by Human Pulp Fibroblasts in Contact with Papacárie and Carisolv Sandra Kalil Bussadoria/Olga Maria Silverio Amanciob/ Manoela Domingues Martinsc/Carolina Cardoso Guedesd/Thays Almeida Alfayae/ Elaine Marcílio Santosf/Cristiane Miranda Françag Purpose: To evaluate the effect of two products for the chemomecanical removal of carious tissue (Papacárie and Carisolv) on human dental pulp fibroblasts and the synthesis of extracellular matrix proteins. Materials and Methods: Fibroblasts were divided into three groups: group 1 (control), group 2 (Papacárie) and group 3 (Carisolv). Collagen I, III, fibronectin and osteonectin were analysed by immunofluorescence and compared among the groups. Results: The groups exhibited similar immunolabeling for vimentin, type I collagen and fibronectin, but were negative for type III collagen. Osteonectin staining was strongly positive in the cells treated with Papacárie and Carisolv and weakly positive in the control group. Conclusion: The findings of the present study showed that Papacárie and Carisolv are not cytotoxic to pulp fibroblast cells. Moreover, these products stimulate fibroblasts to produce osteonectin, likely leading to the formation of dentin matrix. These findings confirm the safe, beneficial use of both gels in minimally invasive techniques. Key words: dental pulp, extracellular matrix proteins, osteonectin, type I collagen, type III collagen Oral Health Prev Dent 2014;1:55-59
Submitted for publication: 25.08.12; accepted for publication: 21.12.12
doi: 10.3290/j.ohpd.a31225
T
he successful outcome of restorative dental treatment requires an understanding of the processes of pulp repair and dentin formation so that treatment decisions can be made in accordance with the natural repair responses of the tooth (Murray et al, 2002). Minimally invasive dentistry involves a ‘systematic respect for the original tissue’, a
Professor, Rehabilitation Sciences Postgraduate Programme, Universidade Nove de Julho, UNINOVE, São Paulo, SP, Brazil.
b
Professor, Paediatrics and Applied Sciences Postgraduate Programme, Universidade Federal de São Paulo University, São Paulo, SP, Brazil.
c
Professor, Department of Oral Pathology, Dental School, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
d
Professor, Department of Paediatric Dentistry, Dental School, Universidade Braz Cubas, Mogi das Cruzes, SP, Brazil.
e
Postgraduate Student, Dental Clinic, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil.
f
Professor, Department of Paediatric Dentistry, Universidade Castelo Branco, São Paulo, SP, Brazil.
g
Professor, Biophotonics Postgraduate Programme, Universidade Nove de Julho, UNINOVE, São Paulo, SP.
Correspondence: Sandra Kalil Bussadori, R. Vergueiro, 235/249 – Liberdade, São Paulo, Brazil 01504-001. Tel: +55-11-3385-9222. Email:
[email protected]
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which implies that the dental professional recognises that an artifact is of less biological value than the original healthy tissue (Ericson, 2004). In the presence of caries, the infected tissue should be removed with the least possible loss of healthy tissue and the most biocompatible materials should be used in restoration. The chemomecanical removal of carious tissue is one of the most widely accepted options in minimally invasive dentistry. This method is based on the dissolution of carious tissue through the application of a natural or synthetic agent which facilitates the elimination of the contaminated tissue with the aid of atraumatic mechanical force, and is followed by restoration with glass-ionomer cement. Carisolv and Papacárie are two such products analysed in the literature (Nadanovsky et al, 2001; Bussadori et al, 2005; Bussadori et al, 2008; Bussadori et al, 2011; Aguirre Aguilar et al, 2012). Carisolv is a red gel composed of three amino acids with different charges that are blended with sodium hypochlorite prior to treatment. Papacárie contains papain and chloramine. Both gels have been studied in vitro and in vivo and have proven to
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be biocompatible in all analyses (Fure et al, 2000; Correa et al, 2007; Martins et al, 2009; Bittencourt et al, 2010). When the dentist chooses a restorative material, it is preferable to use biocompatible materials that, besides being not harmful to the pulp cells, might present additional beneficial effects, such as the induction of dentin synthesis (Costa et al, 2000; de Souza Costa et al, 2001). In its normal state, dental pulp exhibits fibroblasts, odontoblasts and a heterogeneous population of cells, including endothelial, neural, immune and stem cells. The pulp extracellular matrix (ECM) consists of types I, III and IV collagen as well as fibronectin, tenascin, proteoglycans, phospholipids and proteins related to the mineralisation of the tissue, such as osteonectin, osteopontin, osteocalcin and bone sialoprotein (Goldberg and Lasfargues, 1995). Conventional treatment for the removal of carious tissue with low-speed rotary instruments and steel burs can generate additional damage and affect the dentin-odontoblast complex or the pulp (Goldberg and Lasfargues, 1995; Murray et al, 2002). The aim of the present study was to determine whether the gels Papacárie and Carisolv exert an influence on the synthesis of the main proteins of the ECM – types I and III collagen, fibronectin and osteonecti – by pulp fibroblasts.
MATERIALS AND METHODS This study received approval from the ethics committee of the Universidade Federal de São Paulo (Brazil) under process number #39/06. The FP1 cell line – fibroblasts derived from a human third molar germ – was used. FP1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Chemical; St Louis, MO, USA) supplemented with 10% fetal bovine serum (Cultilab; Campinas, SP, Brazil) and 1% antibiotic-antimycotic solution (10,000 units of penicillin, 10 mg of streptomycin and 25 mg of amphotericin B permL in 0.9% sodium chloride; Sigma). The cells were maintained in an incubator at 37°C and a humidified 5% CO2 atmosphere. The cultures were supplied with fresh medium every day. Cells between the fifth and tenth passages were used in all experimental procedures.
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Experimental groups Three groups were established: • Group 1 (G1): control pulp fibroblast cultures grown on a blank coverslip; • Group 2 (G2): cultures of pulp fibroblasts grown on coverslips with Papacárie; • Group 3 (G3): cultures of pulp fibroblasts grown on coverslips with Carisolv. The cells were plated on square glass coverslips, maintained at 37°C in a humid atmosphere containing 5% CO2 for 24 h and then fixed with buffered 4% paraformaldehyde for 1 h. The cells were washed twice in TBS solution (0.15 M NaCl, 20 mM Tris-HCl, 0.05% Tween 20 [Sigma], pH 7.4) and permeabilised with 0.1% Triton X-100 (Sigma) diluted in TBS for 10 min. The cells were then washed twice in both TBS and Milli-Q water and incubated with 5% bovine serum albumin (BSA, Sigma) diluted in PBS for 30 min. Next, the cells were incubated with the primary antibodies, which were diluted in 5% BSA solution in PBSA. The antibodies used were type I collagen (NovoCastra; Newcastle, UK), type III collagen (Dako; Carpinteria, CA, USA), fibronectin (Dako) and osteonectin (NovoCastra) diluted at 1:50 in PBS. Anti-vimentin antibody (Dako) diluted at 1:50 in PBS was used to confirm that the cells analysed were of mesenchymal origin. After incubation with the primary antibody, the cells were washed again with the TBS and Milli-Q water sequence and labeled with the secondary anti-IgG antibodies from sheep-anti-rabbit fluorescein-conjugated secondary antibody (Amersham; Arlington Heights, IL, USA) diluted in PBSA (1:50). All incubations were performed for 60 min at room temperature in the dark. The cells were then washed with the same TBS and Milli-Q water sequence and slides were mounted with Vectashield (Vectar Laboratories; Burlingame, CA, USA). The replacement of the primary antibodies by PBS was used as the negative control. Observations and photographic recording were carried out in a fluorescence microscope (LSM 410 Zeiss; Oberkochen, Germany). For the analysis of immunolabeling, two calibrated observers evaluated the reactivity of each antibody. At the time of examination, the observers had knowledge regarding the antibodies, but were unaware of which samples they were examining. Reactivity scores were established based on the criteria used by Garcia et al (2003), as follows: negative (-), weakly positive (+), positive (++) or strongly positive (+++).
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RESULTS The dental pulp fibroblast cells in all groups exhibited immunolabeling for vimentin, type I collagen, fibronectin and osteonectin (Fig 1) but were negative for collagen type III. The scores are displayed in Table 1. Vimentin exhibited very strong labeling in all groups analysed, demonstrating a reticular network-like appearance in the cytoplasm (Figs 1a to 1c). Fibronectin exhibited intense immunostaining, with a similar distribution pattern in all groups and a reticular network-like appearance (Figs 1d to 1f). Osteonectin was expressed throughout the cytoplasm of the pulp fibroblasts (Figs 1g to 1i). However, weakly positive immunolabeling was observed in the control group, whereas the FP1 cells treated with Papacárie and Carisolv exhibited more intense staining for osteonectin. Type I collagen appeared in the cell as vesicles throughout the cytoplasm (Figs 1j to 1l).
closely resembles an osteoblast than an undifferentiated stem cell. In the present study, stronger osteonectin staining was detected following treatment with Papacárie and Carisolv, which could be VIM
a
VIM
b
c
FN
d
VIM
FN
e
FN
f
ONEC
ONEC
ONEC
DISCUSSION The findings of the present study demonstrate that both Papacárie and Carisolv in contact with pulp fibroblasts stimulate the production of osteonectin and maintain the production of fibronectin and type I collagen at levels similar to those in the control group. This indicates that both gels are not only biocompatible but also beneficial to pulp fibroblasts. Osteonectin, a 32-46 kDa phospho-glycoprotein, is synthesised by human osteoblasts and is the major non-collagen protein in bone tissue (Termine et al, 1981; Romberg et al, 1985). Osteonectin has strong affinity for calcium and hydroxyapatite, likely linking these substances to collagen, and therefore plays a role in mineralisation in vivo (Ingram et al, 1993; Robey, 1996). The detection of osteonectin in cultured pulp fibroblast cells has been analysed previously (Martinez et al, 2000; Garcia et al, 2003). According to those authors, the presence of osteonectin in pulp fibroblasts demonstrates that this type of cell more
g
h
i
Col I
j
Col I
k
Col I
l
Fig 1 Immunofluorescent staining for all proteins studied in all groups. Vimentin [VIM] showed a reticular pattern (a–c); fibronectin [FN] with a network-like appearance in the cytoplasm (d–f); osteonectin [ONEC] revealed a weakly positive reaction in the control group (g) and more intense labeling in the treated groups (h,i); collagen type I [Col I] appeared as a vesicles in the cytoplasm (j-l); (immunofluorescence, original magnification, 1000×).
Table 1 Labeling scores for vimentin, type I and III collagen, fibronectin and osteonectin Groups/antibodies
Vimentin
Type I collagen
Type III collagen
Fibronectin
Osteonectin
G1: Control
+++
+++
–
+++
+
G2: Papacárie
+++
++
–
+++
+++
G3: Carisolv
+++
++
–
+++
+++
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interpreted as stimuli for the formation of mineralised tissue by pulp cells. Type I and type III collagen are the most abundant fibrous proteins found in human dental pulp. Type III collagen has an important function in tissue elasticity (Shuttleworth et al, 1980, van Amerongen et al, 1983), while type I is more commonly found in dense conjunctive tissues and is necessary for the stabilisation of the tissue architecture (Narayanan and Page, 1983). It has been suggested that type I collagen may be either involved in odontoblastic differentiation or a component of predentin secreted by polarised odontoblasts (Abrahao et al, 2006). Type I collagen has been identified in dental papillae during the development of predentin, intertubular dentin and reparative dentin (Martinez and Araujo, 2004). In the present study, type I collagen was expressed in all groups, whereas no immunolabeling of type III collagen occurred. Type III collagen has been detected in pulp tissue in previous in vivo studies, but not observed in cultured pulp cells (Martinez and Araujo, 2004; Abrahao et al, 2006; Martinez et al, 2007). This may be explained by the fact that culture models involve cell growth isolated from other tissues and tension stimuli, which are essential to the formation of type III collagen. On the other hand, the presence of type I collagen may be related to the differentiation of odontoblasts, the formation of dentin matrix and cell stabilisation. The presence of type I collagen and osteonectin in dental pulp fibroblasts indicates that these cells have the potential to participate in the mineral formation process during dentinogenesis, since osteonectin is considered a marker of odontoblast differentiation (Jundt et al, 1987). However, osteonectin alone has no ability to promote mineralisation, as it needs the presence of type I collagen for the precipitation of calcium and phosphate ions (Termine et al, 1981). Fibronectin is an adhesive protein that can be produced by different cell types, such as epithelial, endothelial and stem cells (Ruoslahti, 1981). Fibronectin is deposited in the ECM as a highly insoluble protein associated with a variety of cell functions. In odontogenesis, the presence of this protein in dental papillae is implicated with the migration, polarisation and differentiation of odontoblasts (Lesot et al, 1981; Ruch, 1998). When odontoblasts are aligned, the dental stem cells cease to express fibronectin. However, adult human dental pulp once again exhibits fibronectin, which could be associated with the regulation of cell differentia-
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tion involved in the formation of hard tissue matrices (Thesleff et al, 1987; Lukinmaa et al, 1991; Martinez et al, 2000). FP1 cells, which are the cell line used in the present study, are considered to be the most undifferentiated cells (Garcia et al, 2003). Therefore, the presence of fibronectin could be expected. Martinez et al (2000) did not observe fibronectin in hyalinised, inflammatory areas of human dental pulp. Thus, aggressive injury to the pulp tissue leads to the modification of ECM, with the absence of fibronectin (Garcia et al, 2003). Interestingly, the materials for the removal of carious tissue analysed in the present study cause a low level of injury, allowing greater cell viability and the maintenance of fibronectin labeling, which are characteristics of healthy pulp cells. The presence of vimentin, which in an intermediate filament in the cytoskeleton of pulp fibroblasts, demonstrates that it is a mesenchymal cell line. This marker was used as a control of cell homogeneity (Martinez and Araujo, 2004). Environmental factors alter cellular and extracellular components under different in vitro and in vivo situations (Shiba et al, 1995; Shiba et al, 1998). It is well known that different dental materials or techniques can affect the reparability of the dentinpulp complex by modifying the pulp structure or pulp cell behaviour. The stimulus generated by Papacárie and Carisolv in the present study led the pulp fibroblasts to alter immunolabeling of ECM components involved in dentin formation. The extrapolation of the findings to the clinical setting is not possible due to the dentin barrier and the exposure time of the cells to the products. In clinical practice, this exposure time is only a matter of minutes, whereas exposure time in the present study was 24 h. However, the findings indicate that, besides being biocompatible and not causing cell damage, both gels analysed have the capacity to induce the synthesis of osteonectin. These findings have a considerable potential for clinical applicability.
CONCLUSION The findings of the present study demonstrate that Papacárie and Carisolv are not cytotoxic to pulp fibroblast cells and stimulate the production of osteonectin, which likely leads to the formation of dentin matrix. These results confirm the safe, beneficial use of both gels in minimally invasive techniques.
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REFERENCES 1. Abrahao IJ, Martins MD, Katayama E, Antoniazzi JH, Segmentilli A, Marques MM. Collagen analysis in human tooth germ papillae. Braz Dent J 2006;17:208–212. 2. Aguirre Aguilar AA, Rios Caro TE, Huaman Saavedra J, Franca CM, Fernandes KP, Mesquita-Ferrari RA, et al. [Atraumatic restorative treatment: a dental alternative well-received by children]. Rev Panam Salud Publica 2012;31:148–152. 3. Bittencourt ST, Pereira JR, Rosa AW, Oliveira KS, Ghizoni JS, Oliveira MT. Mineral content removal after Papacarie application in primary teeth: a quantitative analysis. J Clin Pediatr Dent 2010;34:229–231. 4. Bussadori SK, Castro LC, Galvao AC. Papain gel: a new chemo-mechanical caries removal agent. J Clin Pediatr Dent 2005;30:115–119. 5. Bussadori SK, Guedes CC, Bachiega JC, Santis TO, Motta LJ. Clinical and radiographic study of chemical-mechanical removal of caries using Papacarie: 24-month follow up. J Clin Pediatr Dent 2011;35:251–254. 6. Bussadori SK, Guedes CC, Hermida Bruno ML, Ram D. Chemo-mechanical removal of caries in an adolescent patient using a papain gel: case report. J Clin Pediatr Dent 2008;32:177–180. 7. Correa FN, Rocha Rde O, Rodrigues Filho LE, Muench A, Rodrigues CR. Chemical versus conventional caries removal techniques in primary teeth: a microhardness study. J Clin Pediatr Dent 2007;31:187–192. 8. Costa CA, Hebling J, Hanks CT. Current status of pulp capping with dentin adhesive systems: a review. Dent Mater 2000;16:188–197. 9. de Souza Costa CA, Lopes do Nascimento AB, Teixeira HM, Fontana UF. Response of human pulps capped with a selfetching adhesive system. Dent Mater 2001;17:230–240. 10. Ericson D. What is minimally invasive dentistry? Oral Health Prev Dent 2004;2(suppl 1):287–292. 11. Fure S, Lingstrom P, Birkhed D. Evaluation of Carisolv for the chemo-mechanical removal of primary root caries in vivo. Caries Res 2000;34:275–280. 12. Garcia JM, Martins MD, Jaeger RG, Marques MM. Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells. Int Endod J 2003;36: 404–410. 13. Goldberg M, Lasfargues JJ. Pulpo-dentinal complex revisited. J Dent 1995;23:15–20. 14. Ingram RT, Clarke BL, Fisher LW, Fitzpatrick LA. Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity. J Bone Miner Res 1993;8:1019–1029. 15. Jundt G, Berghauser KH, Termine JD, Schulz A. Osteonectin–a differentiation marker of bone cells. Cell Tissue Res 1987;248:409–415. 16. Lesot H, Osman M, Ruch JV. Immunofluorescent localization of collagens, fibronectin, and laminin during terminal differentiation of odontoblasts. Dev Biol 1981;82:371–381. 17. Lukinmaa PL, Mackie EJ, Thesleff I. Immunohistochemical localization of the matrix glycoproteins–tenascin and the ED-sequence-containing form of cellular fibronectin–in human permanent teeth and periodontal ligament. J Dent Res 1991;70:19–26.
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18. Martinez EF, Araujo VC. In vitro immunoexpression of extracellular matrix proteins in dental pulpal and gingival human fibroblasts. Int Endod J 2004;37:749–755. 19. Martinez EF, Araujo VC, Sousa SO, Arana-Chavez VE. TGFbeta1 enhances the expression of alpha-smooth muscle actin in cultured human pulpal fibroblasts: immunochemical and ultrastructural analyses. J Endod 2007;33: 1313–1318. 20. Martinez EF, Machado de Souza SO, Correa L, Cavalcanti de Araujo V. Immunohistochemical localization of tenascin, fibronectin, and type III collagen in human dental pulp. J Endod 2000;26:708–711. 21. Martins MD, Fernandes KP, Motta LJ, Santos EM, Pavesi VC, Bussadori SK. Biocompatibility analysis of chemomechanical caries removal material Papacárie on cultured fibroblasts and subcutaneous tissue. J Dent Child (Chic) 2009;76:123–129. 22. Murray PE, Hafez AA, Windsor LJ, Smith AJ, Cox CF. Comparison of pulp responses following restoration of exposed and non-exposed cavities. J Dent 2002;30:213–222. 23. Murray PE, Windsor LJ, Smyth TW, Hafez AA, Cox CF. Analysis of pulpal reactions to restorative procedures, materials, pulp capping, and future therapies. Crit Rev Oral Biol Med 2002;13:509–520. 24. Nadanovsky P, Cohen Carneiro F, Souza de Mello F. Removal of caries using only hand instruments: a comparison of mechanical and chemo-mechanical methods. Caries Res 2001;35:384–389. 25. Narayanan AS, Page RC. Connective tissues of the periodontium: a summary of current work. Coll Relat Res 1983;3:33–64. 26. Robey PG. Vertebrate mineralized matrix proteins: structure and function. Connect Tissue Res 1996;35:131–136. 27. Romberg RW, Werness PG, Lollar P, Riggs BL, Mann KG. Isolation and characterization of native adult osteonectin. J Biol Chem 1985;260:2728–2736. 28. Ruch JV. Odontoblast commitment and differentiation. Biochem Cell Biol 1998;76:923–938. 29. Ruoslahti E. Fibronectin. J Oral Pathol 1981;10:3–13. 30. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, et al. Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol 1998;174: 194–205. 31. Shiba H, Nakamura S, Shirakawa M, Nakanishi K, Okamoto H, Satakeda H, et al. Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells. Dev Biol 1995;170:457–466. 32. Shuttleworth CA, Berry L, Wilson N. Collagen synthesis in rabbit dental pulp fibroblast cultures. Arch Oral Biol 1980;25:201–205. 33. Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR. Osteonectin, a bone-specific protein linking mineral to collagen. Cell 1981;26:99–105. 34. Thesleff I, Mackie E, Vainio S, Chiquet-Ehrismann R. Changes in the distribution of tenascin during tooth development. Development 1987;101:289–296. 35. van Amerongen JP, Lemmens IG, Tonino GJ. The concentration, extractability and characterization of collagen in human dental pulp. Arch Oral Biol 1983;28:339–345.
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