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Immunohistochemical Expression of TGF-β1 and Osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

Abstract

The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250–425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100–70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.

Dental Pulp; Transforming Growth Factors; Osteonectin; Tissue Engineering

Introduction

During tooth development, a precise temporal-spatial expression of bioactive glycoprotein-based molecules, known as growth factors (GF), regulates the crosstalk between the epithelial and mesenchymal germ layers,11. Nör JE. Tooth regeneration in operative dentistry. Oper Dent. 2006;31(6):633-42. doi:10.2341/06-000
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,22. Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med.. 2004;15(1):13-27.doi:10.2485/jhtb.13.55
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leading to odontoblast differentiation.33. Ruch JV, Lesot H, Bègue-Kirn C. Odontoblast differentiation. Int J Dev Biol. 1995;39(1):51-68. The odontoblasts form a terminally specialized hard tissue. These cells are responsible for secreting the dentin extracellular matrix (DECM) both during the odontogenesis and pulp-dentin complex (PDC) repair.44. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28(2):77-92. doi:10.1016/S0300-5712(99)00047-0
https://doi.org/10.1016/S0300-5712(99)00...
,55. Tabatabaei FS, Ai J, Jafarzadeh Kashi TS, Khazaei M, Kajbafzadeh AM, Ghanbari Z. Effect of dentine matrix proteins on human endometrial adult stem-like cells: in vitro regeneration of odontoblasts cells. Arch Oral Biol. 2013;58(7):871-9. doi:10.1016/j.archoralbio.2013.01.013
https://doi.org/10.1016/j.archoralbio.20...
After the complete dentin mineralization, the GF becomes fossilized in the dentin matrix, making the tissue a rich source of bioactive molecules.66. Smith JG, Smith AJ, Shelton RM, Cooper PR. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components. Exp Cell Res. 2012;318(18):2397-406. doi:10.1016/j.yexcr.2012.07.008
https://doi.org/10.1016/j.yexcr.2012.07....
,77. Schmalz G, Smith AJ. Pulp development, repair, and regeneration: challenges of the transition from traditional dentistry to biologically based therapies. J Endod. 2014;40(4 Suppl):S2-5. doi:10.1016/j.joen.2014.01.018
https://doi.org/10.1016/j.joen.2014.01.0...

Because of the secretory activity of odontoblasts, the PDC can react to the external stimuli (caries, trauma) by evoking defense responses.88. Simon SR, Berdal A, Cooper PR, Lumley PJ, Tomson PL, Smith AJ. Dentin-pulp complex regeneration: from lab to clinic. Adv Dental Res. 2011;23(3):340-5. doi:10.1177/0022034511405327
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Although moderate carious lesions stimulate the secretory activity of odontoblasts (reactionary dentinogenesis),44. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28(2):77-92. doi:10.1016/S0300-5712(99)00047-0
https://doi.org/10.1016/S0300-5712(99)00...
,99. Couve E, Osorio R, Schmachtenberg O. Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries. J Dent Res. 2014;93(8):788-93. doi:10.1177/0022034514539507
https://doi.org/10.1177/0022034514539507...
deep cavity preparation or severe carious lesions may lead to the partial destruction of the odontoblastic layer (reparative dentinogenesis). In the reparative dentinogenesis, a population of undifferentiated cells is recruited from the pulp core to the injury site where they differentiate into odontoblast-like cells, starting the deposition of reparative dentin.1010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
Ca(OH)2-based materials have been applied in dentistry to preserve the pulp vitality by inducing a dentin bridge formation.1111. Hilton TJ. Keys to clinical success with pulp capping: a review of the literature. Oper Dent. 2009;34(5):615-25. doi:10.2341/09-132-0
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,1212. Chisini LA, Conde MCM, Correa MB, Dantas RVF, Silva AF, Pappen FG et al. Vital pulp therapies in clinical practice: findings from a survey with dentist in Southern Brazil. Braz Dent J. 2015;26(6):566-71. doi:10.1590/0103-6440201300409
https://doi.org/10.1590/0103-64402013004...
These materials, because of their high pH, solubilize the DECM, promoting mobilization and recruitment of fossilized GFs.1010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
,1313. Tomson PL, Grover LM, Lumley PJ, Sloan AJ, Smith AJ, Cooper PR. Dissolution of bio-active dentine matrix components by mineral trioxide aggregate. J Dent. 2007;35(8):636-42. doi:10.1016/j.jdent.2007.04.008
https://doi.org/10.1016/j.jdent.2007.04....
Dentin-derived GFs are necessary for the differentiation of dental pulp stem cells (DPSC) into odontoblasts.1414. Demarco FF, Casagrande L, Zhang Z, Dong Z, Tarquinio SB, Zeitlin BD et al. Effects of morphogen and scaffold porogen on the differentiation of dental pulp stem cells. J Endod. 2010;36(11):1805-11. doi:10.1016/j.joen.2010.08.031
https://doi.org/10.1016/j.joen.2010.08.0...
,1515. Conde MC, Chisini LA, Demarco FF, Nör JE, Casagrande L, Tarquinio SB. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int Endod J. 2016;49(6):543-50. doi:10.1111/iej.12489
https://doi.org/10.1111/iej.12489...
The available data reinforce the hypothesis that the molecular and cellular processes involved in PDC healing and regenerative events recapitulate the odontogenesis.44. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28(2):77-92. doi:10.1016/S0300-5712(99)00047-0
https://doi.org/10.1016/S0300-5712(99)00...
,1616. Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis: embryonic events as a template for dental tissue repair? Crit Rev Oral Biol Med 2001;12(5):425-37. doi:10.1177/10454411010120050501
https://doi.org/10.1177/1045441101012005...

The transforming growth factor-β (TGF-β) family is an important modulator of odontoblast activity, responsible for many molecular events during tooth development and repair.1717. Sloan AJ, Perry H, Matthews JB, Smith AJ. Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth. Histochem J. 2000;32(4):247-52. doi:10.1023/A:1004007202404
https://doi.org/10.1023/A:1004007202404...
During caries development, the immunoexpression of the isoform 1 (TGF-β1) in odontoblastic cells is enhanced compared with sound teeth.1717. Sloan AJ, Perry H, Matthews JB, Smith AJ. Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth. Histochem J. 2000;32(4):247-52. doi:10.1023/A:1004007202404
https://doi.org/10.1023/A:1004007202404...
Furthermore, TGF-β1 acts as a potent chemotactic factor for STRO-1-sorted undifferentiated cells.1818. Mathieu S, Jeanneau C, Sheibat-Othman N, Kalaji N, Fessi H, About I. Usefulness of controlled release of growth factors in investigating the early events of dentin-pulp regeneration. J Endod. 2013;39(2):228-35. doi:10.1016/j.joen.2012.11.007
https://doi.org/10.1016/j.joen.2012.11.0...
This has been demonstrated by examining the mineralization process coordinated by the dental pulp cells1919. Liu J, Jin T, Chang S, Ritchie HH, Smith AJ, Clarkson BH. Matrix and TGF-beta-related gene expression during human dental pulp stem cell (DPSC) mineralization. In Vitro Cell Dev Biol Anim. 2007;43(3-4):120-8. doi:10.1007/s11626-007-9022-8
https://doi.org/10.1007/s11626-007-9022-...
and the effect of the controlled release of TGF-β1 on the pulp cell proliferation and migration. ON is a multifunctional non-collagenous glycoprotein, involved in cell morphogenesis, migration, and differentiation.2020. Cheng L, Sage EH, Yan Q. SPARC fusion protein induces cellular adhesive signaling. PLoS One. 2013;8(1):e53202. doi:10.1371/journal.pone.0053202
https://doi.org/10.1371/journal.pone.005...
This protein has been implicated in orchestrating the interactions between cells and its substrates,2121. Salonen J, Domenicucci C, Goldberg HA, Sodek J. Immunohistochemical localization of SPARC (osteonectin) and denatured collagen and their relationship to remodelling in rat dental tissues. Arch Oral Biol. 1990;35(5):337-46. doi:10.1016/0003-9969(90)90180-I
https://doi.org/10.1016/0003-9969(90)901...
coordinating cell adhesion, proliferation, and matrix synthesis and turnover.2222. Motamed K. SPARC (osteonectin/BM-40). Int J Biochem Cell Biol. 1999;31(12):1363-6. doi:10.1016/S1357-2725(99)00090-4
https://doi.org/10.1016/S1357-2725(99)00...

Because the molecular and cellular processes responsible for dentinogenesis are recapitulated during PDC repair,44. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28(2):77-92. doi:10.1016/S0300-5712(99)00047-0
https://doi.org/10.1016/S0300-5712(99)00...
,1616. Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis: embryonic events as a template for dental tissue repair? Crit Rev Oral Biol Med 2001;12(5):425-37. doi:10.1177/10454411010120050501
https://doi.org/10.1177/1045441101012005...
it might be useful to compare the expression of the molecules responsible for both events. The aim of our study was to compare the immunoexpression of TGF-β1 and ON in tooth slice/scaffold (TS/S)-regenerated pulp-like tissue and in Ca(OH)2-repaired pulps.

Methodology

Chemicals

The cell culture medium and reagents were supplied by Invitrogen (Grand Island, NY, USA). All the other reagents were obtained from Sigma-Aldrich Chemical Co. (St. Louis, USA), except for phosphate-buffered saline (PBS), which was obtained from Mediatech, Inc. (Herndon, USA), and Poly-L-lactic acid, from Boehringer (Ingelheim, Germany).

Cells

DPSCs, provided by Dr. Songtao Shi were cultured at 37°C in 5% CO2 in low-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. To conduct the experiments (Figure 1), we used cells from 4th–6th passage.

Figure 1
Flowchart of planned protocol with reference to the previous material and method (tooth slice/scaffold and clinical procedure).

TS/S preparation

Non-carious human third molars were obtained, after obtaining informed consent and following the approved institutional review board protocol, from young patients (17–23 years old) at the Oral Surgery Clinic (University of Michigan School of Dentistry). The teeth were transversely sectioned at the cervical region. We used a diamond blade at a low speed under cooling with sterile PBS to obtain 1-mm thick tooth slices.2323. Gonçalves SB, Dong Z, Bramante CM, Holland GR, Smith AJ, Nör JE. Tooth slice-based models for the study of human dental pulp angiogenesis. J Endod. 2007;33(7):811-4. doi:10.1016/j.joen.2007.03.012
https://doi.org/10.1016/j.joen.2007.03.0...
The pulp tissue was carefully removed leaving an empty space in the tooth slice. Sodium chloride particles (250–425 µm) were sieved and used to fill the empty pulp chamber, and PLLA solubilized in chloroform (5%) was dropped over the salt particles. TS/S was stored overnight to permit the PLLA polymerization. Subsequently, the salt was washed out by submersion in distilled water for 24 h (the water was changed 3 times).

Preparation for seeding

Scaffolds were sterilized using ethanol of descending grades (100–70°) and washed with PBS. All scaffolds were treated for 1 min with 10% EDTA (pH = 7.2) and washed again with PBS immediately before cell seeding.

Clinical procedure

As a positive control, we used histological sections provided by Dr. Evandro Piva.2424. Piva E, Tarquínio SB, Demarco FF, Silva AF, Araújo VC. Immunohistochemical expression of fibronectin and tenascin after direct pulp capping with calcium hydroxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(4):e66-71. doi:10.1016/j.tripleo.2006.01.015
https://doi.org/10.1016/j.tripleo.2006.0...
Briefly, the human pulp tissue was exposed by a preparation of class I cavities (carbide bur #245) under refrigeration, with cooled PBS. The exposure sites were cleaned using PBS, and the hemorrhage was controlled using sterile cotton pellets. The exposed pulp tissue was capped with calcium hydroxide powder (Biodinâmica, Ibiporã, Brazil). Calcium hydroxide cement (Dycal; Dentsply, Petrópolis, Brazil) was applied over the powder. Cavities were sealed with reinforced zinc oxide–eugenol cement (IRM; Dentsply). Teeth were extracted after 90 days, and the apical root portion was sectioned to improve the formalin penetration inside the pulp.

In Vivo culture of DPSCs

DPSCs, 6 × 1055. Tabatabaei FS, Ai J, Jafarzadeh Kashi TS, Khazaei M, Kajbafzadeh AM, Ghanbari Z. Effect of dentine matrix proteins on human endometrial adult stem-like cells: in vitro regeneration of odontoblasts cells. Arch Oral Biol. 2013;58(7):871-9. doi:10.1016/j.archoralbio.2013.01.013
https://doi.org/10.1016/j.archoralbio.20...
, were re-suspended in a 1:1 DMEM: Growth Factor Reduced Matrigel, seeded in the PLLA TS/Ss or control scaffolds and cultured (at 37°C and 5% CO2) for 30 min to allow cell attachment. Then, each TS/S was implanted in the dorsum of 5–7-week-old male immunodeficient mouse (CB-17 SCID; Charles River, MA). After 28 days, the implants were retrieved from the dorsum, fixed in 10% buffered formalin at 4°C for 24 h, and demineralized with 10% formic acid at 4°C until the dentin offered no resistance to cutting with a blade (10–15 days). Histological sections (5 mm thick) were prepared from non-carious human third molars and from TS/S as described previously.1414. Demarco FF, Casagrande L, Zhang Z, Dong Z, Tarquinio SB, Zeitlin BD et al. Effects of morphogen and scaffold porogen on the differentiation of dental pulp stem cells. J Endod. 2010;36(11):1805-11. doi:10.1016/j.joen.2010.08.031
https://doi.org/10.1016/j.joen.2010.08.0...
All experiments in animals were performed following the guidelines reviewed and approved by the University of Michigan Review Board.

Histological sample preparation

The samples were immersed in 5% formic acid until complete decalcification and washed for 48 h in deionized water. The samples were fixed, hemi-sectioned, and inserted in paraffin. Two slices (3 µm) were obtained from each histological sample (n = 3). Slices were stained with hematoxylin and eosin.

Immunohistochemical analysis

Paraffin blocks were cut into five samples of 3 μm thickness. Each sample was placed on silanized glass (Pró-cite). TGF-β1 antibody (Dakocytomation; Dako, Carpinteria, CA) was used at 1:100 dilution for 18 h at 4°C and ON antibody (1:350; Lab Vision, Fremont, CA), for 18 h at 4°C. We used the streptavidin-biotin method for immunohistochemical analysis. After washing, samples underwent antigenic recuperation treatment with 0.5% pepsin, pH 1.8, for 30 min at 37°C, to reestablish the antigenic sites and break the crosslinking. The slices were washed in tap water, followed by two rinses with distilled water. Unspecific protein blocking was performed by immersion in 10% skim milk solution for 30 min. After the blocking, the slices were washed in water followed by two baths in a Tris-HCl buffer solution. The samples were then incubated with primary antibodies following the manufacturer’s instructions and washed in Tris-HCl buffer solution. Incubation with tertiary serum and tertiary complex—Kit LSAB (Dako Corporation, California) — was then performed (both for 30 min). Distilled water and Tris buffer were used to wash the samples. The samples were placed in diaminobenzidine chromogen (AEC, Dakocytomation) for 1 min, counterstained with Mayer hematoxylin for 8 min, and then washed.

Results

Regenerated pulp-like tissues

Twenty-eight days after implantation, DPSCs generated a tissue inside TS/S, with morphological characteristics of a pulp tissue. This tissue, resembling repaired dental pulp, was found at the scaffold filling the pulp chamber (Figures 2 and 3).

Figure 2
Immunolocalization of ON in regenerated (tooth slice/scaffolds) and repaired (Ca(OH)2) pulps. D: Dentin; P: Pulp; PD: Predentin; CH: Ca(OH)2; TS: Tooth slice; DPD: Demineralized (pre)dentin; PLTECM: Pulp-like tissue extracellular matrix.

Figure 3
Immunolocalization of TGF-β1 in regenerated (tooth slice/scaffolds) and repaired (Ca(OH)2) pulps. D: Dentin; P: Pulp; PD: Predentin; CH: Ca(OH)2; TS: Tooth slice; DPD: Demineralized predentin; PLTECM: Pulp-like tissue extracellular matrix.

Immunohistochemical features of regenerated and repaired tissues

The expression of TGFβ-1 and ON was similar in the repaired and regenerated tissues. In both cases, the ECM staining showed a fibrillar pattern (Figure 2 and 3), reflecting the affinity of TGF-β1 and ON antibodies to the pulp ECM. The pre-dentin from both TS/S and Ca(OH)2-repaired pulp was strongly stained. However, the cellular elements (mainly fibroblasts) were not immunoreactive to the antibodies evaluated here.

Discussion

To analyze the features shared during the repair and regeneration in the PDC, we examined the immunoexpression of TGF-β1 and ON under both conditions.2424. Piva E, Tarquínio SB, Demarco FF, Silva AF, Araújo VC. Immunohistochemical expression of fibronectin and tenascin after direct pulp capping with calcium hydroxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(4):e66-71. doi:10.1016/j.tripleo.2006.01.015
https://doi.org/10.1016/j.tripleo.2006.0...
The results showed that the ECM and predentin, both from regenerated and repaired tissues, reacted with TGF-β1 and ON antibodies. During dentinogenesis, a set of GFs orchestrates the epithelial-mesenchymal interactions, leading to odontoblastic differentiation of primitive cells from dental papilla.2525. Smith AJ. Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. J Dental Educ. 2003;67(6):678-89. After DECM mineralization, such GFs are fossilized inside the dentin in their latent form.2626. Sloan AJ, Smith AJ. Stimulation of the dentine-pulp complex of rat incisor teeth by transforming growth factor-beta isoforms 1-3 in vitro. Arch Oral Biol. 1999;44(2):149-56. doi:10.1016/S0003-9969(98)00106-X
https://doi.org/10.1016/S0003-9969(98)00...
Thus, dentin becomes a reservoir of latent biomolecules.2525. Smith AJ. Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. J Dental Educ. 2003;67(6):678-89. When solubilizing agents such as Ca(OH)21010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
or EDTA1515. Conde MC, Chisini LA, Demarco FF, Nör JE, Casagrande L, Tarquinio SB. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int Endod J. 2016;49(6):543-50. doi:10.1111/iej.12489
https://doi.org/10.1111/iej.12489...
are placed over the dentin walls, the fossilized GFs are released to trigger molecular cascades responsible for repair and regeneration.66. Smith JG, Smith AJ, Shelton RM, Cooper PR. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components. Exp Cell Res. 2012;318(18):2397-406. doi:10.1016/j.yexcr.2012.07.008
https://doi.org/10.1016/j.yexcr.2012.07....
It is interesting to note that the released GFs are diffused into the pulp tissue to participate in the cell migration and differentiation.2525. Smith AJ. Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. J Dental Educ. 2003;67(6):678-89.

The fibrillar pattern observed in the immunohistochemical analysis is probably due to the presence of collagen and some non-collagen proteins such as fibronectin.2727. Mjör IA, Sveen OB, Heyeraas KJ. Pulp-dentin biology in restorative dentistry. Part 1: normal structure and physiology. Quintessence Int. 2001;32(6):427-46. Fibronectin mediates the binding of signaling molecules to ECM, playing a critical role during interactions between ECM and the cells.44. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28(2):77-92. doi:10.1016/S0300-5712(99)00047-0
https://doi.org/10.1016/S0300-5712(99)00...
The linking between GFs and ECM seems crucial to cell activation.66. Smith JG, Smith AJ, Shelton RM, Cooper PR. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components. Exp Cell Res. 2012;318(18):2397-406. doi:10.1016/j.yexcr.2012.07.008
https://doi.org/10.1016/j.yexcr.2012.07....
It is a key event prolonging their action during PDC regeneration and repair.66. Smith JG, Smith AJ, Shelton RM, Cooper PR. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components. Exp Cell Res. 2012;318(18):2397-406. doi:10.1016/j.yexcr.2012.07.008
https://doi.org/10.1016/j.yexcr.2012.07....
In addition, the activity of GFs is dose-dependent. Thus, an optimal concentration of GFs is needed to trigger a specific biological cascade. GFs might cause cell damage in concentrations higher than a specific dose required.11. Nör JE. Tooth regeneration in operative dentistry. Oper Dent. 2006;31(6):633-42. doi:10.2341/06-000
https://doi.org/10.2341/06-000...
It is possible that the presence of TGF-β1 and ON in the region of predentin and pulp(-like) tissues is due to both the dissolution and activation of the GF, throughout the collagen fibers and some non-collagen proteins such as fibronectin.2828. Bronckers AL, Lyaruu DM, Wöltgens JH. Immunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis. Connecet Tissue Res. 1989;22(1-4):65-70. doi:10.3109/03008208909114121
https://doi.org/10.3109/0300820890911412...

The ultimate goal of pulp regeneration strategies is to reconstitute a normal tissue continuum at the pulp-dentin border, regulating tissue-specific processes of secondary and/or tertiary dentinogenesis.2929. Demarco FF, Conde MC, Cavalcanti BN, Casagrande L, Sakai VT, Nör JE. Dental pulp tissue engineering. Braz Dent J. 2011;22(1):3-13. doi:10.1590/S0103-64402011000100001
https://doi.org/10.1590/S0103-6440201100...
Therefore, the newly formed pulp-like tissue must contain odontoblast-like cells capable of secreting predentin in the host organism.2323. Gonçalves SB, Dong Z, Bramante CM, Holland GR, Smith AJ, Nör JE. Tooth slice-based models for the study of human dental pulp angiogenesis. J Endod. 2007;33(7):811-4. doi:10.1016/j.joen.2007.03.012
https://doi.org/10.1016/j.joen.2007.03.0...
,2929. Demarco FF, Conde MC, Cavalcanti BN, Casagrande L, Sakai VT, Nör JE. Dental pulp tissue engineering. Braz Dent J. 2011;22(1):3-13. doi:10.1590/S0103-64402011000100001
https://doi.org/10.1590/S0103-6440201100...
Here, DPSCs were seeded in TS/S and implanted in the dorsa of immunodeficient mice. TS/S is an effective tool for the investigation of DPSC proliferation and differentiation.1414. Demarco FF, Casagrande L, Zhang Z, Dong Z, Tarquinio SB, Zeitlin BD et al. Effects of morphogen and scaffold porogen on the differentiation of dental pulp stem cells. J Endod. 2010;36(11):1805-11. doi:10.1016/j.joen.2010.08.031
https://doi.org/10.1016/j.joen.2010.08.0...
,1515. Conde MC, Chisini LA, Demarco FF, Nör JE, Casagrande L, Tarquinio SB. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int Endod J. 2016;49(6):543-50. doi:10.1111/iej.12489
https://doi.org/10.1111/iej.12489...
When the dentin walls of TS/S are treated with EDTA, fossilized GFs are released, and cell differentiation is induced.3030. Laurent P, Camps J, About I. Biodentine(TM) induces TGF-ß1 release from human pulp cells and early dental pulp mineralization. Int endod J. 2012;45(5):439-48. doi:10.1111/j.1365-2591.2011.01995.x
https://doi.org/10.1111/j.1365-2591.2011...
DPSCs cultured on TS/S under the conditions described here are able to express the three putative odontoblastic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1), and matrix extracellular phosphoglycoprotein (MEPE).

During repair, the stimulus reduces blood flow, inducing the GF (such as TGF-β1) release from the ECM.1010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
Samples from healthy human pulps capped with calcium hydroxide for 90 days were also evaluated here. ON was present in the predentin and in the repaired region. Calcium hydroxide can stimulate mineralization, acting like osteodentin, because of its ability to solubilize DECM and release GFs.1010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
ON has been found in predentin and in intertubular dentin,3131. Reichert T, Störkel S, Becker K, Fisher LW. The role of osteonectin in human tooth development: an immunohistological study. Calcif Tissue Int. 1992;50(5):468-72. doi:10.1007/BF00296779
https://doi.org/10.1007/BF00296779...
which we also observed here. TGF-β1 is incorporated into DECM.3232. Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S, Baylink DJ. Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin. J Bone Miner Res. 1990;5(7):717-23. doi:10.1002/jbmr.5650050708
https://doi.org/10.1002/jbmr.5650050708...
After DECM demineralization, TGF-β1 induces stem cell migration to the damaged site and their odontoblastic differentiation.1010. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ. The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials. 2006;27(14):2865-73. doi:10.1016/j.biomaterials.2005.12.020
https://doi.org/10.1016/j.biomaterials.2...
During the ECM formation,3333. Shao MY, Cheng R, Wang FM, Yang H, Cheng L, Hu T. ß-Catenin and Rho GTPases as downstream targets of TGF-ß1 during pulp repair. Cell Biol Int. 2011;35(2):105-9. doi:10.1042/CBI20100114
https://doi.org/10.1042/CBI20100114...
TGF-β1 inhibits ECM degradation.3434. Overall CM, Wrana JL, Sodek J. Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-beta. J Biol Chem. 1989;264(3):1860-9. It is also an important mediator of ECM remodeling,3636. Farges JC, Romeas A, Melin M, Pin JJ, Lebecque S, Lucchini M, et al. TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer. J Dent Res. 2003;82(8):652-6. inducing actin fiber formation.3535. Salinas PC. Modulation of the microtubule cytoskeleton: a role for a divergent canonical Wnt pathway. Trends Cell Biol. 2007;17(7):333-42. The isoforms of TGF, -β1 and -β3, stimulate the PDC response during the formation of tertiary dentin. The isoform β3 also effectively induces the odontoblastic differentiation during PDC regeneration.2626. Sloan AJ, Smith AJ. Stimulation of the dentine-pulp complex of rat incisor teeth by transforming growth factor-beta isoforms 1-3 in vitro. Arch Oral Biol. 1999;44(2):149-56. doi:10.1016/S0003-9969(98)00106-X
https://doi.org/10.1016/S0003-9969(98)00...

ON is strongly expressed during dentinogenesis.33. Ruch JV, Lesot H, Bègue-Kirn C. Odontoblast differentiation. Int J Dev Biol. 1995;39(1):51-68. Because of its affinity to hydroxyapatite, ON is involved in molecular cascades determining the hydroxyapatite formation and the crystal stabilization.2222. Motamed K. SPARC (osteonectin/BM-40). Int J Biochem Cell Biol. 1999;31(12):1363-6. doi:10.1016/S1357-2725(99)00090-4
https://doi.org/10.1016/S1357-2725(99)00...
Thus, this protein plays a central role during reactionary or sclerotic dentinogenesis by modulating the dentin tubule obliteration under pathological conditions.3737. Magloire H, Romeas A, Melin M, Couble ML, Bleicher F, Farges JC. Molecular regulation of odontoblast activity under dentin injury. Adv Dental Research. 2001;15:46-50. ON is present in the odontoblast layer;3838. Takano-Yamamoto T, Takemura T, Kitamura Y, Nomura S. Site-specific expression of mRNAs for osteonectin, osteocalcin, and osteopontin revealed by in situ hybridization in rat periodontal ligament during physiological tooth movement. J Histochem Cytochem. 1994;42(7):885-96. its distribution seems to be restricted to the unmineralized predentin of the intertubular dentin and the lamina limitans.3939. Papagerakis P, Berdal A, Mesbah M, Peuchmaur M, Malaval L, Nydegger J, et al. Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth. Bone. 2002;30(2):377-85. When the odontoblast layer is destroyed, ON is released and stimulates the proliferation of a fraction of pulp cells, which differentiate into odontoblasts to form the reparative dentin.4040. Shiba H, Uchida Y, Kamihagi K, Sakata M, Fujita T, Nakamura S, et al. Transforming growth factor-beta1 and basic fibroblast growth factor modulate osteocalcin and osteonectin/SPARC syntheses in vitamin-D-activated pulp cells. J Dent Res. 2001;80(7):1653-9.

Conclusion

On the basis of our data, we could conclude that the GF evaluated here acts in a similar manner during the pulp repair and regeneration. TGF-β1 is involved in attracting the progenitor pulp cells and the stabilization of ECM, where the stem cells attach. ON is expressed by odontoblast-like cells, indicating their differentiation. The regenerative events in PDC follow a pattern of TGF-β1 and ON expression similar to the pattern seen during the repair process.

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    » https://doi.org/10.1042/CBI20100114
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  • 38
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  • 39
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  • 40
    Shiba H, Uchida Y, Kamihagi K, Sakata M, Fujita T, Nakamura S, et al. Transforming growth factor-beta1 and basic fibroblast growth factor modulate osteocalcin and osteonectin/SPARC syntheses in vitamin-D-activated pulp cells. J Dent Res. 2001;80(7):1653-9.

Publication Dates

  • Publication in this collection
    2016

History

  • Received
    14 Mar 2016
  • Reviewed
    30 Apr 2016
  • Accepted
    06 June 2016
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