The role of integrin β1 in human dental pulp cell adhesion on laminin and fibronectin

VoL 85 No. 3 March 1998

ENDODONTICS

Editor: R i c h a r d E. Walton

The role of integrin [51 in human dental pulp cell adhesion on laminin and fibronectin Qiang Zhu, DDS, PhD, a Kamran E. Safari, DMD, MEd, b and Larz S. W. Spangberg, DDS, PhD, ¢ Farmington, Conn. THE UNIVERSITYOF CONNECTICUTHEALTHCENTER

Objective. This study is to identify the expression of integrin [31 in human dental pulp cells and the role of integrin [31 in pulp cell adhesion on extracellular matrix protein laminin and fibronectin. Study design. Immunoblot detection of integrin 131 in human dental pulp cells was with the use of monoclonal anti-J31 antibody. Dental pulp cell adhesion assay on extracellular matrix protein laminin and fibronectin and blocking cell adhesion was performed with monoclonal anti-131 antibody. Result. Integrin [31 was identified in human dental pulp cells. Pulp cells adhered and spread on both laminin and fibronectin, Monoclonal anti-131 antibody inhibited human dental pulp cells adhesion on laminin but not on fibronectin. Conclusions. lntegrin ffl was expressed on human dental pulp cells and mediated cell adhesion on laminin. Human dental pulp cells also adhered on fibronectin but the adhesion was not regulated by [31 integrin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998:85;314-8)

Dental ectomesenchymal cells from neural crest give rise to odontoblasts during tooth development. The differentiation of odontoblasts requires specific interactions between the inner dental epithelium and the dental mesenchyme. 1,2 The cell-cell interactions are mediated by transcription and growth factors, extracellular matrix proteins, and cell surface receptors. 3,4 Odontoblasts produce dentin that surrounds the dental pulp tissue in the mature tooth. After tooth formation, the pulp is able to form reparative dentin after direct pulp capping with calcium hydroxide 5,6 or by induction with bone morphogenetic protein. 7 The exact mechanisms by which dental pulp cells differentiate into odontoblasts is unknown. Studies have indicated the extracellular matrix glycoprotein fibronectin is involved in the odontoblast differentiation and initiation of reparative dentinogenesis during pulp cells response to calcium hydroxide: 8,9 aAssistant Professor, Department of Restorative Dentistry and Endodontology, School of Dental Medicine. bAssociate Professor and Program Director, Department of Restorative Dentistry and Endodontology,School of Medicine. Cprofessor and Head, Department of Restorative Dentistry and Endodontology, School of Medicine. Received for publication June 9, 1997; returned for revision Oct. 9, 1997; acceptedfor publicationNov. 2, 1997. Copyright © 1998 by Mosby,Inc. 1079-2104/98/$5.00 + 0 7/15187515

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An in vitro study has shown the regulatory role of bone morphogenetic proteins on gene expression of fibronecfin and the differentiation of pulp cells into preodontoblasts. 1° Fibronectin is a multifunctional extracellular glycoprotein that mediates cell attachment and migration. It plays an important role in many biologic processes, such as cell adhesion and differentiation, wounding healing, embryogenesis, tumor cell invasion, and metastasis. 11 Laminin is another well-studied noncollagenous glycoprotein of the extracellular matrix. It performs numerous biologic activities, including stimulating cell adhesion, migration, and differentiation and promoting neurite outgrowth. 12 Cells bind fibronectin and laminin mainly by means of cell surface receptor integrins. 13 Fibronectin and laminin provide a multitude of signals to the contact cells through integrin, whereas the cell-extracellular matrix interaction influences the behavior of cells and the architecture of tissues, Study of dental pulp cell surface receptor integfin will help to understand the effect of fibronecfin and laminin on dentinogenesis as well as the differentiation of dental pulp cells to odontoblasts. There is no published report on integrins of dental pulp cells and their role in the interaction between dental pulp cells and extracellular matrix protein laminin and fibronectin. The purpose of this study is to identify

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Volume 85, Number 3 the expression of 131integrin in human dental pulp cells and to determine the role of integrin ~1 in pulp cell adhesion on laminin and fibronectin.

MATERIAL AND METHOD Cell culture Human pulp cells were grown from pulp tissue. Normal human pulp was obtained from extracted impacted third molars. The culture medium was Eagle's MEM (Life Technologies, Inc. Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc. Logan, Utah) and 1% antibiotic/ antimycotic cocktail (300 U/ml penicillin, 300 ~tg/ml streptomycin, 5 gg/ml amphotericin B). The pulp tissue was minced to 1 mm 3 pieces, placed in 25 cm 2 culture flasks and incubated under standard cell culture conditions (37°C, 100% humidity, 95% air and 5% carbon dioxide). When the cell growth from pulp tissue had reached confluence, the cells were detached with 0.05% trypsin (Life Technologies, Inc.) and subcultured in 75 cm 2 culture flasks (Becton Dickinson Labware, Franklin Lakes, N.J.). The cells were used at subculture levels 3 through 10.

Cell adhesion assay Cell adhesion assays were essentially performed as described earlier. 14 The 96-well microtiter plates (Dynatech Laboratories Inc. Chantilly, Va.) were coated with 5 Ixg/well laminin or fibronectin (Life Technologies, Inc.) in phosphate-buffered saline solution (PBS). Albumin-coated wells were used as control. The protein-coated wells were blocked with 10 mg/ml albumin in PBS for 10 minutes at room temperature and then rinsed three times with PBS before cell adhesion assays. The human pulp cells were seeded in triplicate at 2.0 × 104 cells per well in serum-free minimal essential medium and allowed to incubate for 1 hour at 37 ° C under standard culture conditions. Then the incubation medium was dumped out of the plates. All wells were washed three times with PBS. The adherent cells were fixed with 4% paraformaldehyde for 20 minutes and then stained with 1% toluidine blue for 2 hours at room temperature. Cells were visually examined and photographed with an inverted microscope. The effect of anti-[~1 integrin antibody (Life Technologies, Inc.) on human dental pulp cells adhesion was performed by preincubating the cells with 1:100 dilution of anti-131 antibody or hybridoma supernatants (Sigma, St. Louis, Mo.) as control for 15 minutes at room temperature before the assay.

Electrophoresis and immunoblot Human pulp ceils were washed with PBS three times and lysed in lysis buffer containing 150 mM sodium

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48Fig. 1. Immunoblot analysis of integrin ~1 with monoclonal anti-[~1 antibody. The numbers on the left are standard molecular weight (kDa). The expression of integrin [31is shown on all three (lanes 1-3) tested strains of human dental pulp cells. chloride, 50 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS and protease inhibitors (1 mM PMSF, 2 gg/ml aprotinin, 2 gg/ml leupeptin, 0.4 gg/ml antipain, 2 mg/gl benzamidine, 1 gg/ml chymostatin and 1 gg/ml pepstatin). SDS-polyacrylamine gel electrophoresis was carried out according to the method of Laemmli15; 5% to 15% polyacrylamine gel was used. Electrophoretically separated proteins were blotted onto supported nitrocellulose (Schleicher and Schuell, Keene, N.H.) with the method of Towbin et al. 16 The nitrocellulose filter was incubated with 1:1000 rat anti-human131 antibody (PharMingen, San Diego, Calif.) overnight at 4 ° C and then incubated with 1:2000 horseradish peroxidase conjugated rabbit anti-rat IgG secondary antibody (Sigma) for 2 hours at room temperature. Visualization of imrnunoreactive proteins was achieved using the ECL detection kit (Amersham Life Science, Arlington Heights, Ill.).

RESULTS The immunoblot experiment showed ~1 integrin in all tested human dental pulp cells from three donors by using anti-~l monoclonal antibody (Fig. 1). The attachment experiments showed human dental pulp cells attached and spread both on laminin (Fig. 2, A) and fibronectin (Fig. 2, B). The pulp cells had a more sprinkled spreading on laminin than fibronectin. In the controls, human dental pulp cells did not attach to bovine

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Fig. 2. A, Human dental pulp cells adhering and spreading on laminin. Note different morphologies that cells attain on laminin as compared to fibronectin. Cell spreading on laminin is more sprinkled than cell spreading on fibronectin (Fig. 2, B). (Original magnification xl00.) B, Adhesion and spreading of human dental pulp cells on fibronectin. (Original magnification xl00.) C, Human dental pulp cells do not adhere on bovine serum albumin. (Original magnification ×100.)

serum albumin (Fig. 2, C). Although the monoclonal anti-~l antibody had no influence on cell adhesion on fibronectin, the cell shape was changed to smaller and irregular when anti-[~! antibody was present (Fig. 3, A). The antibody inhibited pulp cell adhesion on laminin (Fig. 3, B). In the control group, mouse hybridoma IgG supernatants did not affect adhesion and spreading of pulp cells on either fibronectin or laminin (Fig. 3, C).

DISCUSSION Integrins are a family of heterodimeric transmembrane glycoproteins that bind many components of extracellular matrix. Integrin consists of an o~ and a I~ subunit. At least 16 different t~ subunits and 8 different I~ subunits have been identified with more than 20 different members of integrins. They mediate cell attachment to extracellular matrix and cell-cell adhesive interactions. Cell-extracellular matrix interaction through integrins regulates cell growth, adhesion, differentiation, and cell morphology.J1,13 A recent study 17 shows that human osteoblasts express specific integrins, syn-

thesize different amounts of extracellular matrix proteins, and have different morphologic characteristics on different implant materials. The expression of different integrins could determine the different responses of osteoblasts. 17 This study shows integrin I]1 is expressed in human dental pulp cells and provides evidence that integrin also plays an important role in biologic functions of dental pulp cells. Laminin and fibronectin are major extracellular matrix proteins that stimulate cell adhesion, spreading, migration, and differentiation. We showed that human dental pulp cells adhere and spread both on laminin and fibronectin. In controls, the cells failed to adhere on bovine serum albumin. Laminin and fibronectin induce human dental pulp cells adhesion and spreading. In another study fibronectin and laminin have been shown to enhance gingival cell attachment to dental implant surfaces) 8 The different morphologies that cells attain on laminin compared with fibronectin have also been observed in rat glioblastoma cells.19 When anti-Ill monoclonal antibody was used try to block cell adhesion,

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Fig. 3. A, Human dental pulp cells attach and spread on fibronectin in presence of monoclonal anti-~l antibody. Note that cell shape is irregular compared to cell appearance on fibronectin. (Original magnification MOO.) B, Monoclonal anti-]31 antibody inhibits human dental pulp cells adhesion on laminin. (Original magnification xlOO.) C, Mouse hybridoma IgG has no effect on human dental pulp cells adhesion and spreading on laminin. (Original magnification x 1OO.)

pulp cells failed to adhere on l a m i n i n but not on fibronectin. Thus, human dental pulp cells apparently adhere on l a m i n i n mainly through [31 integrin. Even though the anti-[31 antibody does not influence pulp cells attachment on fibronectin, it alters the cell shape. This suggests integrin [31 is involved in human dental pulp cells spreading on fibronectin. Integrin 91 has a biologic role in both adhesion and spreading of human dental pulp cells. The results of these findings will help further the understanding of the interactions between pulp cells and extracellular matrix proteins. Further areas of study could focus on the expression of integrins on human pulp cells during reparative dentin formation after treatment of pulp exposures as well as the application of extracellular matrix proteins on resected rootends and retrofilling materials during apical surgery to stimulate cell attachment and wound healing. We thank Dr. Marvin L. Tanzer for his support and help of this study. We thank Dr. Bruce Rutherford for his advice and generously providing one strain of human dental pulp cells.

REFERENCES 1. Ruch JV. Tooth crown morphogenesisand cytodifferentiations: candid questions and critical comments. Connect Tissue Res 1995;32:1-8. 2. MinaM, KollarEJ. The inductionof odontogenesisin non-dental mesenchymecombin~-dwith early murine mandibulararch epithelium.Arch Oral Bk.; 1987;32:123-7. 3. ThesleffI, NieminenR Tooth morphogenesisand cell differentiation. Curt Opin Cell Biol 1996;8:844-50. 4. Maas R, Bei M. The geneticcontrolof early tooth development. Crit Rev Oral Biol Med 1997;8:4-39. 5. CvekM. A clinicalreport on partialpulpotomyand cappingwith calcium hydroxide in permanent incisors with complicated crown fracture. J Endod 1978;4:232-7. 6. Schroder U. Effects of calciumhydroxide-containingpulp-capping agents on pulp cell migration,proliferation,and differentiation. J Dent Res 1985;64:541-8. 7. Rutherford RB, Wahle J, Tucker M, Rueger D, Charette M. Inductionof reparative dentin formationin monkeysby recombinant human osteogenic protein-1.Archs Oral Biol 1993;38: 571-6. 8. Tziafas D, Panagiotakopoulos N, Komnenou A. Immunolocalizationof fibronectinduringthe early response of dog dental pulp to demineralizeddentineor calciumhydroxide-containing cement.Archs Oral Biol 1995;40:23-31. 9. Yoshiba K, Yoshiba N, Nakamura H, Iwaku M, Ozawa H. Immonolocalizationof fibronectin during reparative dentino-

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genesis in human teeth after pulp capping with calcium hydroxide. J Dent Res 1996;75:1590-7. Nakashima M, Nagasawa H, Yamada Y, Reddi AH. Regulatory role of transforming growth factor-J3, bone morphogenetic prorein-2, and protein-4 on gene expression of extracellnlar matrix proteins and differentiation of dental pulp cells. Dev Biol 1994;162:18-28. Aota S-I, Yamada KM. Fibronecfin and cell adhesion: specificity of integfin-ligand interaction. Adv Enzymol Relat Areas Mol Biol 1995;70:1-21. Tanzer ML, Chandrasekaran S, Dean JW, Giniger MS. Role of laminin carbohydrates on cellular interactions. Kidney Intl 1993;34:66-72. Hynes RO. Integfins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11-25. White TK, Zhu Q, Tanzer ML. Cell surface calreficulin is a putative mannoside lectin which triggers mouse melanoma cell spreading. J Biol Chem 1995;270:15926-9. Laemrnli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-8.

16. Towbin S, Staehlin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76: 4350-4. 17. Gronowicz G, McCarthy MB. Response of human osteoblasts to implant materials: integrin-mediated adhesion. J Orthop Res 1996;14:878-87. 18. Dean JW, Culbertson KC, Dangelo AM. Fibronectin and laminin enhance gingival cell attachment to dental implant surfaces in vitro. J Oral Maxillofac Implants 1995;10:721-8. 19. Wewer UM, Engvall E. Laminins. Methods Enzymol 1994; 245:85-104.

Reprint requests: Qiang Zhu, DDS, PhD Assistant Professor Department of Restorative Dentistry and Endodontology School of Dental Medicine The University of Connecticut Health Center Farmington, CT 06030-1715

CALL FOR LETTERSTO THE EDITOR A separate and distinct space for Letters to the Editor was established by Larry J. Peterson, editor in chief of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics in his Editorial in the January 1993 issue. Dr. Peterson also encouraged brief reports on interesting observations and new developments to be submitted to appear in this letters section as well as Letters commenting on earlier published articles. Please submit your letters and brief reports for inclusion in this section. Information for authors for the Journal appears in this issue of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and

Endodontics. We look forward to hearing from you.