Abstracts

Introduction

Odontoblasts are post-mitotic cells responsible for primary, secondary and reactionary dentinogenesis. After differentiation, they are no longer able to multiply, which limits the regenerative capability of the pulp ^{( 1}1Demarco FF, Conde MC, Cavalcanti BN, Casagrande L, Sakai VT, Nor JE. Dental pulp tissue engineering. Braz Dent J 2011;22:3-13. ^{, 2}2Baldissera EZ, Silva AF, Gomes AP, Etges A, Botero T, Demarco FF, et al.. Tenascin and fibronectin expression after pulp capping with different hemostatic agents: preliminary study. Braz Dent J 2013;24:188-193. ⁾. Damage to dental pulp induces inflammatory responses with different degrees, corresponding to the nature and strength of injury ⁽²⁾2Baldissera EZ, Silva AF, Gomes AP, Etges A, Botero T, Demarco FF, et al.. Tenascin and fibronectin expression after pulp capping with different hemostatic agents: preliminary study. Braz Dent J 2013;24:188-193.. In pathological conditions, such as mild carious dentine lesions, odontoblastic activity is stimulated to elaborate reactionary dentine ⁽³⁾3Bjorndal L, Mjor IA. Pulp-dentin biology in restorative dentistry. Part 4: Dental caries - characteristics of lesions and pulpal reactions. Quintessence Int 2001;32:717-736.. However, in case of acute injuries by deep caries cavity preparations odontoblasts may be lost and dental pulp may undergo necrosis ⁽⁴⁾4Heyeraas KJ, Sveen OB, Mjor IA. Pulp-dentin biology in restorative dentistry. Part 3: Pulpal inflammation and its sequelae. Quintessence Int 2001;32:611-625..

Traditionally, when pulp tissue undergoes necrosis, conventional endodontic therapy (CET) must be performed to remove the necrotic tissues ⁽⁵⁾5Friedman S, Mor C. The success of endodontic therapy - healing and functionality. J Calif Dent Assoc 2004;32:493-503.. Despite the good results observed following endodontic therapy, in some situations CET may not be the first choice treatment ⁽⁴⁾4Heyeraas KJ, Sveen OB, Mjor IA. Pulp-dentin biology in restorative dentistry. Part 3: Pulpal inflammation and its sequelae. Quintessence Int 2001;32:611-625..

Pulp necrosis in immature permanent teeth (IPT) occurs after trauma or untreated carious lesions ⁽⁶⁾6Nor JE. Tooth regeneration in operative dentistry. Oper Dent 2006;31:633-642.. CET turns the root brittle, as IPT present discontinued root development with thin dentinal walls associated to the need of getting rid of dentin inside the pulp chamber, favoring the occurrence of root fracture in case of a new trauma. Also, there is an additional challenge to obtain an appropriated apical seal in IPT by applying CET ⁽⁶⁾. Currently, necrosed immature permanent teeth are subjected to multiple-visit apexification with Ca(OH)₂-based materials to induce formation of an apical mineralized barrier ^{( 6}6Nor JE. Tooth regeneration in operative dentistry. Oper Dent 2006;31:633-642. ^{, 7}7Moreno-Hidalgo MC, Caleza-Jimenez C, Mendoza-Mendoza A, Iglesias-Linares A. Revascularization of immature permanent teeth with apical periodontitis. Int Endod J 2014;47:321-331. ⁾. Despite the good prognosis, the long-term Ca(OH)₂-based apexification presents a series of drawbacks ⁽⁸⁾8Dantas RV, Conde MC, Sarmento HR, Zanchi CH, Tarquinio SB, Ogliari FA, et al.. Novel experimental cements for use on the dentin-pulp complex. Braz Dent J 2012;23:344-350., such as multiple treatment appointments, probable recontamination of the root canal system during treatment and increased brittleness of the root dentin, which increases the risk of future cervical root fractures ⁽⁷⁾7Moreno-Hidalgo MC, Caleza-Jimenez C, Mendoza-Mendoza A, Iglesias-Linares A. Revascularization of immature permanent teeth with apical periodontitis. Int Endod J 2014;47:321-331..

In this sense, the development of a new dental pulp by tissue engineering has produced increased interest ⁽¹⁾1Demarco FF, Conde MC, Cavalcanti BN, Casagrande L, Sakai VT, Nor JE. Dental pulp tissue engineering. Braz Dent J 2011;22:3-13.. The three key elements of tissue engineering are responsive cells, knows as stem cells, morphogens or growth factors and scaffolds. Scaffolds are three-dimensional structures used to support and guide the ingrowth of cells, acting as an extracellular matrix (ECM) analogue ⁽⁹⁾9Langer R, Vacanti JP. Tissue engineering. Science1993;260:920-926.. Dental pulp stem cells (DPSC) are multipotent stem cells able to differentiate into a wide range of tissues. Moreover, DPSC exhibit after odontoblastic stimulation ⁽¹⁰⁾10Demarco 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;36:1805-1811., the expression of genes, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), related to the differentiation of stem cells towards an odontoblast-like cell phenotype ⁽¹¹⁾11Goldberg 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;15:13-27..

Stem cell behavior relies on the scaffold's spatial properties such as porosity, pore size and void fraction ⁽¹²⁾12Lawrence BJ, Madihally SV. Cell colonization in degradable 3D porous matrices. Cell Adh Migr 2008;2:9-16.. Scaffold's pore size has been shown to affect the cell attachment, proliferation, migration, morphology and gene expression ^{( 10}10Demarco 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;36:1805-1811. ^{, 13}13Santana BP, Paganotto GF, Nedel F, Piva E, de Carvalho RV, Nor JE, et al.. Nano/microfiber scaffold for tissue engineering: physical and biological properties. J Biomed Mater Res A 2012;100;3051-3058. ⁾. The smaller the scaffold's pore size the greater the specific surface area for cell attachment on its surface; however, the cell migration can be hindered ^{( 12}12Lawrence BJ, Madihally SV. Cell colonization in degradable 3D porous matrices. Cell Adh Migr 2008;2:9-16. ⁾. Additionally, it has direct influence on the diffusion of nutrients and removal of waste into the scaffold, resulting in necrotic regions within the construct ⁽¹⁴⁾14Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010;31:461-466.. On the other hand, the larger the scaffold's pores the easier the traffic of nutrients, gas diffusion and metabolic residue removal. However, it reduces the relative surface for cell attachment and a possible specific site as the integrin ^{( 14}14Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010;31:461-466. ⁾. Therefore, it is imperative for the future of dental tissue engineering to analyze physical characteristics of materials used to mimic the extracellular matrix.

The most favorable pore size range depends on the nature of the material and cell used in the constructs ^{( 15}15Zeltinger J, Sherwood JK, Graham DA, Mueller R, Griffith LG. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng 2001;7:557-572. ^{, 16}16Salem AK, Stevens R, Pearson RG, Davies MC, Tendler SJ, Roberts CJ, et al.. Interactions of 3T3 fibroblasts and endothelial cells with defined pore features. J Biomed Mater Res 2002;61:212-217. ⁾. In poly-L-lactic acid (PLLA) scaffolds, vascular smooth muscle cells bind to a smaller pore range size (63-150 µm), while fibroblasts bind to a wider range (38-150 µm) ⁽¹⁵⁾15Zeltinger J, Sherwood JK, Graham DA, Mueller R, Griffith LG. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng 2001;7:557-572.. In addition, for bone tissue engineering a range of pore sizes comprising 100-150 and 150-200 µm have shown substantial bone ingrowth, while a smaller range (75-100 µm) results in the formation of an non-mineralized osteoid tissue ^{( 14}14Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010;31:461-466. ^{, 17}17Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials2005;26:5474-5491. ⁾. Besides, DPSC are able to attach and proliferate into PLLA scaffolds with a pore size ranging from 250-425 µm, prepared in a tooth slice/scaffold (TS/S) model and produce a de novo pulp-like tissue ^{( 10}10Demarco 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;36:1805-1811. ^{, 18}18Casagrande L, Demarco FF, Zhang Z, Araujo FB, Shi S, Nor JE. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res 2010;89:603-608. ⁾. Therefore, this study aims to evaluate the influence of PLLA scaffold's pore size on the proliferation and differentiation of DPSC using the TS/S model.

Material and Methods

Chemicals

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, MO, USA), except for the phosphate buffered saline (PBS), which was purchased from Mediatech, Inc. (Herndon, VA, USA) and the poly-L-lactic acid from Boehringer (Ingelheim, Germany).

Cells

Dental pulp stem cells (DPSC), gently provided by Dr. Songtao Shi (Dental Biology Unit, Craniofacial Skeletal Diseases Branch, NIH, Bethesda, MD, USA) were isolated by standard protocol (Gronthos et al., 2000). The cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) low glucose-containing 10% fetal bovine serum and 1% penicillin/streptomycin solution and incubated at 37 °C in 5% CO₂. During the experiments, cells from passage 4-6^th were used.

Porogen Production and Tooth Slice/Scaffold (TS/S) Preparation

Sodium chloride (salt) was sieved using metallic sieves producing two particle sizes: 150-250µm and 251-425µm. After sieving, the salt was stored in Petri dishes until test. At the oral surgery clinic in the Dental School, University of Michigan, USA - noncarious human third molars were extracted from healthy and young patients (17-23 years old) after signing an informed consent and under an approved institutional review board protocol. Residual soft tissues were removed with a periodontal scalpel, and the dental surfaces were wiped with 70% ethanol. Teeth were transversely sectioned at the cervical region with a diamond blade at low speed under cooling with sterile phosphate-buffered saline (PBS) to obtain 1-mm thick tooth slices. The pulp tissue was thoroughly removed with sterile forceps and the dentin was conditioned for 1 min with ethylenediamine tetraacetic acid (EDTA) and washed again with PBS ⁽¹⁸⁾18Casagrande L, Demarco FF, Zhang Z, Araujo FB, Shi S, Nor JE. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res 2010;89:603-608.. Sodium chloride was sieved (150-250 µm and 250-425mm) and filled the pulp chamber. The tooth slices (1-mm thick) had their void pulp chamber (Fig. 1) filled with salt particles. The PLLA scaffolds were produced into TS/S using the solvent-casting/particulate leaching technique - SC/PL ⁽¹⁹⁾19Lu L, Peter SJ, Lyman MD, Lai HL, Leite SM, Tamada JA, et al.. In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials2000;21:1837-1845.. Briefly, PLLA (Boehringer Ingelheim, Germany) was dissolved (5% w/v) with chloroform, and the mixture was carefully dropped over the salt. After PLLA polymerization, the salt was leached with distilled water for 24 h.

Figure 1.
Diagram depicting the overall scheme for the model systems used here. A: 1-mm thick tooth slices were prepared from the cervical region of human third molars. B: Standardization of measures of the pulp chamber space into the tooth slices.

Cell Seeding into TS/S

DPSC at the subconfluent stage (80%) were detached with 0.25% trypsin-EDTA and 1x10⁵ cells in a 20 µL cell suspension (DMEM) were seeded inside each 6 TS/S and placed in a 24-well plate. Immediately after seeding, samples were placed in incubator (37 °C in 5% CO₂) for 1 h to allow initial cell attachment. Then, 500 µL DMEM was added in each well, and the medium was changed every other day.

Gene Expression

To evaluate the relative gene expression was used the reverse transcriptase - polymerase chain reaction (RT-PCR). After 21 days in culture, three scaffolds per condition were pooled and total ribonucleic acid (RNA) was isolated and purified using the Trizol^(r) system (Invitrogen) as recommended by the manufacturer ⁽¹⁰⁾10Demarco 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;36:1805-1811.. As control, odontoblasts were scraped from freshly extracted human third molars and RNA was isolated ⁽²⁰⁾20Conde MC, Nedel F, Campos VF, Smith AJ, Nor JE, Demarco FF, et al.. Odontoblast RNA stability in different temperature-based protocols for tooth storage. Int Endod J 2012;45:266-272.. Total RNA (0.2 µg), from odontoblast and TS/S was used to perform the RT-PCR. The human-specific sense and antisense primers were designed according to published cDNA sequences of GenBank (Table 1). Three independent experiments were performed to verify the reproducibility of the results.

Relative Cell Density (RCD)

RCD in the TS/S was checked after 3, 7, 14 and 21 days using the WST-1 dye ⁽²¹⁾21Nedel F, Soki FN, Conde MC, Zeitlin BD, Tarquinio SB, Nor JE, et al.. Comparative analysis of two colorimetric assays in dental pulp cell density. Int Endod J 2011;44:59-64.. Briefly, at each time point, 20 µL WST-1 were mixed with 200 µL of fresh DMEM medium and incubated during 1 h to develop the reaction. Then, an aliquot of 100 µL was removed from each well and placed into a 96-well plate in order to evaluate the optical density of the formazan salts produced by the viable cells using an enzyme-linked immunosorbent assay (ELISA) multiplate reader (TECAN, Genius) with a wavelength filter of 450 nm.

Statistical Analysis

Data from RCD were submitted to statistical analysis using two-way ANOVA followed by Tukey test using Sigmastat 2.0 software (SSPS, Chicago, IL, USA) and the significance level was set at p<0.05. For each condition/time, triplicates were performed and the experiments were repeated at least three times.

Results

Relative Cell Density

Both pore sizes evaluated here produced similar RCD in the different test time-points. However, it was observed on the fourteenth day that RCD was higher in the scaffolds with larger porosities (Fig. 2). The cells in both scaffolds had a continuous growth up to 21 days, when a slight decrease was observed, indicating that the cells reached the confluence or differentiation.

Figure 2.
WST-1 analysis demonstrating the RCD rates produced on the two scaffolds. Day 14 (p<0.05).

Gene Expression (RT-PCR)

DPSC seeded in the TS/S with both pore sizes exhibited after 21 days the expression of all putative odontoblast markers (DSPP, DSP-1 and MEPE), similar to control. House-keeping gene was also expressed by control and experimental groups (Fig. 3).

Figure 3.
RT-PCR analysis showing that the DPSC were able to express the odontoblast markers after have being seeded for 21 days into both scaffolds. OD=Odontoblastic RNA; S<=Scaffolds with minor pore size; S>=Scaffolds with larger pore size. GAPDH: Housekeeping gene.

Discussion

Both pore size ranges evaluated here produced similar effects on the DPSC proliferation and odontoblastic gene expression - DSPP, DMP-1 and MEPE ⁽¹¹⁾11Goldberg 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;15:13-27.. Considering the lack of information about this specific topic in the dental tissue engineering, it was chosen to evaluate the PLLA scaffold pore size over the proliferation and differentiation ability of DPSC.

To carry out the experiments was used the TS/S method, which is well established as an efficient model to evaluate the proliferation and differentiation rates of stem cells from dental tissues ^{( 10}10Demarco 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;36:1805-1811. ^{, 18}18Casagrande L, Demarco FF, Zhang Z, Araujo FB, Shi S, Nor JE. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res 2010;89:603-608. ⁾. Furthermore, Demarco et al. ⁽¹⁰⁾10Demarco 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;36:1805-1811. has shown that the TS/S is useful to evaluate physical properties of the scaffolds. Comparing two PLLA scaffolds with different porous formats (spherical x cubic), they were able to show that both porogens provided an adequate environment for DPSC proliferation and differentiation (positive for MEPE, DMP1 and DSPP). Thus, it was tested whether the same pore size (251-425 µm) tested before ⁽¹⁰⁾, and a smaller one could had some influence on the DPSC proliferation and differentiation, since it was previously reported that smaller porous sizes increased the specific surface area for cell attachment, which influences directly the RCD ⁽²²⁾22El-Backly RM, Massoud AG, El-Badry AM, Sherif RA, Marei MK. Regeneration of dentine/pulp-like tissue using a dental pulp stem cell/poly(lactic-co-glycolic) acid scaffold construct in New Zealand white rabbits. Aust Endod J;2008;34:52-67..

To evaluate RCD, the 4-[3-(4-Iodophenyl)-2-(4-nitrophe-nyl)-2H5tetrazolio]-1,3-benzene disulfonate, WST-1 test was applied, which has been reported as an effective tool to determine the proliferation rates of DPSC ⁽²¹⁾21Nedel F, Soki FN, Conde MC, Zeitlin BD, Tarquinio SB, Nor JE, et al.. Comparative analysis of two colorimetric assays in dental pulp cell density. Int Endod J 2011;44:59-64.. Tetrazolium salt-based methods, such as WST-1 are simple and efficient procedures to analyze the DPSC proliferation based on cell metabolism. In such methods, the specific tetrazolium salt, a yellow reagent, is metabolically reduced by the cell machinery into a bright purple colored formazan salt end-product ⁽²³⁾23Berridge M, Tan A, McCoy K, Wang R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica1996;4:14-19.. Thus, a spectrophotometer is used to quantify the intensity of the color, producing numerical data (absorbance) that correlate with the number of metabolically active cells ⁽²¹⁾. Here, WST-1 test showed an increase in RCD (Fig. 2) with time, until DPSC reached confluence or differentiation occurred in both evaluated pore ranges. This shows that the different evaluated scaffolds were sufficiently permeable enabling cell growth, migration and nutrition. Nevertheless, a significant difference in RCD was observed at day 14. In such specific time point, the DPSC seeded in the scaffolds with larger pores (251-425 µm) showed a higher proliferation rate than those seeded in the scaffolds with smaller pores.

From these data, it was hypothesized that higher pore size (HPS) allowed a faster DPSC migration and proliferation, which allowed the DPSC reach the confluence faster than cells seeded into scaffolds with smaller pores (SPS). Murphy et al. ⁽¹⁴⁾14Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010;31:461-466. comparing a range of scaffolds with different pore sizes, showed that larger pore size is able to improve cellular adhesion and infiltration into scaffolds up to 7 days post-seeding. However, those results were obtained in a series of collagen-glycosaminoglycan (CG) scaffolds. It is well established that cells can discriminate subtle changes in the ECM and it affects their behavior ⁽¹²⁾12Lawrence BJ, Madihally SV. Cell colonization in degradable 3D porous matrices. Cell Adh Migr 2008;2:9-16.; therefore, the results should be compared with caution. In addition, a consistent slowdown was observed in total RCD at day 21 into HPS, similar to the one observed in the SPS. In an attempt to explain it, two hypotheses were raised: 1) cells had reached confluence into TS/S with large pores after day 14; 2) higher cell densities, provided by larger mean pores, inhibited the proliferation by cell-cell contact and it was accentuated by the crowding space, which induces a morphologic alteration in cells and could induce cell death ⁽²⁴⁾24Lanosa XA, Colombo JA. Cell contact-inhibition signaling as part of wound healing processes in brain. Neuron Glia Biol 2008;4:27-34.. It is interesting to point out that scaffold pore size affects other stem cells' behavior behind RCD. In this context, deep analyses should be carried out because other variables can be responsible for cell density decrease.

In the present study, it was not possible to observe a statistical difference in RCD when DPSC were seeded into the both tested scaffolds. Therefore, it was hypothesized that the theoretically faster cell proliferation provided by the larger pores overlaid the benefits provided by the major specific area in the scaffolds with smaller pores ⁽¹⁴⁾14Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials2010;31:461-466..

The scaffolds' volume should be taken into account to evaluate the stem cell proliferation based on TE principles ^{( 1}1Demarco FF, Conde MC, Cavalcanti BN, Casagrande L, Sakai VT, Nor JE. Dental pulp tissue engineering. Braz Dent J 2011;22:3-13. ^{, 10}10Demarco 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;36:1805-1811. ^{, 13}13Santana BP, Paganotto GF, Nedel F, Piva E, de Carvalho RV, Nor JE, et al.. Nano/microfiber scaffold for tissue engineering: physical and biological properties. J Biomed Mater Res A 2012;100;3051-3058. ⁾. When slices from different teeth are used, clearly the pulp chamber volume will vary. In the TS/S model, the scaffold is produced in a direct way inside the pulp chamber from human teeth ⁽¹⁰⁾10Demarco 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;36:1805-1811.. The anatomical variation inherent for each tooth could produce a bias when carrying out the evaluation of cell proliferation by applying colorimetric tests, like WST-1, since this tool is influenced by the number of cells in the scaffold ⁽²¹⁾21Nedel F, Soki FN, Conde MC, Zeitlin BD, Tarquinio SB, Nor JE, et al.. Comparative analysis of two colorimetric assays in dental pulp cell density. Int Endod J 2011;44:59-64.. To minimize such bias, the teeth used in the present study were standardized. To be included here all teeth should have similar characteristics (erupted third molar from young patients). Using a digital caliper, the largest rays of the pulp chamber were measured in two axes (Fig. 1). Thus, a constant measures were obtained - the tooth slice thickness (1 mm) and the two major widths of pulp chamber - and these values were standardized, allowing the maximum of 10% variation between the chamber sizes among specimens in the tested groups.

To assess the differentiation of DPSC in odontoblast-like genotype was evaluated the relative gene expression of DSPP, DMP-1 and MEPE using the RT-PCR based on a previous study ⁽²⁰⁾20Conde MC, Nedel F, Campos VF, Smith AJ, Nor JE, Demarco FF, et al.. Odontoblast RNA stability in different temperature-based protocols for tooth storage. Int Endod J 2012;45:266-272.. After 21 days, DPSC in both HPS and SPS were able to express the three putative odontoblast markers into TS/S as previously reported in other studies ^{( 10}10Demarco 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;36:1805-1811. ^{, 18}18Casagrande L, Demarco FF, Zhang Z, Araujo FB, Shi S, Nor JE. Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res 2010;89:603-608. ^{, 25}25Sakai VT, Zhang Z, Dong Z, Neiva KG, Machado MA, Shi S, et al.. SHED differentiate into functional odontoblasts and endothelium. J Dent Res 2010;89:791-796. ⁾. RT-PCR showed that the evaluated pore sizes did not influence the odontoblast markers expression. Using the TS/S, the process of PLLA scaffold degradation should be considered; due to lactic acid release and a possible acidification of the environment would impair the expression of DSPP, DMP-1 and MEPE. However, in a previous work ⁽¹⁰⁾10Demarco 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;36:1805-1811. was evaluated the expression of the above-mentioned genes in vitro and in vivo, and the results obtained showed that the PLLA scaffolds did not impair the odontoblast markers expression and formation of pulp-like tissue in vivo. As matter of fact, scaffold degradation is a prerequisite to accomplish differentiation of stem cells towards odontoblastic-like genotype ⁽¹⁰⁾. The products of PLLA scaffold degradation, instead of disturbing the stem cell differentiation contributed to the dentin solubilization of growth factors, which in turn have acted as morphogenic agents to direct DPSC or SHED differentiation towards an odontoblast-like genotype.

Within the limitations of this study, it was possible to conclude that the two ranges of pore sizes evaluated in this study provided a favorable environment for DPSC proliferation and differentiation, with overall similar results between them.

Every knowledge generated about biomaterials (scaffolds) coupled with advances in clinical research, will provide tools to translate regenerative pulp therapies into clinical dental practice.

Acknowledgements

The study was funded by grant R01-DE21410 from the NIH/NIDCR (JEN). The authors would also like to ackowledge the Brazilian Government (# 080/2009/DPB-CAPES) for the scholarship provided to the first author (MCMC).

Publication Dates

History