Highlights
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Our study provides a comparison between rough implant texture and a ceramic one.
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3D rotating cell culture were carried out by seeding DPSCs on dental implants.
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Both surfaces promote cell adhesion, proliferation and differentiation.
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We note greater cell proliferation in TiUnite, earlier differentiation in TriVent.
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A slower mineralization process may be more conducive to longer stability.
Abstract
Objective
The major challenge for contemporary dentistry is restoration of missing teeth; currently, dental implantation is the treatment of choice in this circumstance. In the present study, we assessed the interaction between implants and Dental Pulp Stem Cells (DPSCs) in vitro by means of 3D cell culture in order to better simulate physiological conditions.
Methods
Sorted CD34 + DPSCs were seeded onto dental implants having either a rough surface (TriVent) or one coated with a ceramic layer mimicking native bone (TiUnite). We evaluated preservation of DPSC viability during osteogenic differentiation by an MTT assay and compared mineralized matrix deposition with SEM analysis and histological staining; temporal expression of osteogenic markers was evaluated by RT-PCR and ELISA.
Results
Both surfaces are equally biocompatible, preserve DPSC viability, stimulate osteogenic differentiation, and increase the production of VEGF. A slight difference was observed between the two surfaces concerning the speed of DPSC differentiation.
Significance
Our study of the two implant surfaces suggests that TriVent, with its roughness, is capable of promoting cell differentiation a bit earlier than the TiUnite surface, although the latter promotes greater cell proliferation.
1
Introduction
Edentulism can be considered the basis of chronic esthetic and functional deterioration of the oral cavity due to progressive resorption of the maxillary bony structures, resulting in ptosis of the overlying soft tissues. Importantly, tooth loss produces progressive deficiency in the normal functions of the mouth, such as chewing, swallowing, and phonation . The main causes of tooth loss are traumatic events, endodontic pathologies, neglecting caries, and, especially, periodontal disease. Tooth loss represents a major challenge for contemporary dentistry, which recognizes dental implantation as the treatment of choice in this situation in order to restore masticatory function and the esthetics of the patients .
Implant design has a key role in osteointegration, a direct structural and functional connection between living bone and the surface of the implant . An implant is integrated, and hence capable of supporting the load placed upon it, when all the gaps at the bone–implant interface are filled with newly synthesized bone tissue. This process is carried out by Mesenchymal Stem Cells (MSCs) . MNCs have self-renewal properties and are capable of differentiating into many cellular lineages upon appropriate induction . Immediately after implant insertion, local blood vessel growth allows the recruitment of migratory MSCs to the surgical site and the implant’s surface. The cells then proliferate and differentiate into mature osteoblasts responsible for bone matrix deposition, a process essential for perfect implant integration. Bone formation around the implant depends on the level of differentiation of the MSCs. This process is largely affected by the nature of the implant: in fact, studies have reported that chemical, mechanical, and topographic characteristics of the implant surface influence all events involved in bone formation at the bone–implant interface, including cell adhesion, proliferation, differentiation, and matrix deposition . For these reasons, many implant textures – obtained through anodization, calcium phosphate coating, chemical or biological modification, sandblasting, etc. – have been designed and studied in vitro and in vivo over the last decades in an attempt to improve osteointegration .
Today, titanium and its alloys are recognized as the material of choice in implant dentistry because of their excellent biologic and biomechanical properties, their biocompatibility, and the ease with which different textures can be produced . Roughness is the most-studied characteristic of implant texture, and it has been reported that this feature is important in enhancing osteoblast differentiation, with increased roughness maximizing osteoinduction . However, reports of the effects of titanium surface roughness on cell differentiation are contrasting .
Therefore, we decided to investigate how implant texture affects the early phases of osteodifferentiation in vitro. The study was conducted with Dental Pulp Stem Cells (DPSCs), since they are easily extracted with high efficiency from dental pulp ; moreover, DPSCs are largely employed in tissue engineering because of their capability to differentiate into many cells types, such as mature osteoblasts , and due to their ability to generate a three-dimensional (3D) bone tissue in vitro . Moreover, these cells have been recently investigated also with regards to their specific migratory capabilities in relationship with other stem cells .
In addition, these cells are naturally delegated to dental tissue repair: in fact, DPSCs have been successfully employed in bone repair in vitro and in vivo .
2
Materials and methods
2.1
Dental implants
The dental implants used in this study were Tri-Vent, purchased from TRI Dental Implants Int AG (Baar, Switzerland), and TiUnite, purchased from Nobel Biocare (Nobel Biocare Goteborg, Sweden). Tri-Vent is a sandblasted, acid-etched surface created by blasting the implant surface under pressure; TiUnite is composed of a slightly rough titanium oxide layer covered with a phosphorus coat conferring it a ceramic-like property rich in micropores.
2.2
Cell extraction and 2D culture
Mesenchymal stem cells were obtained by the extraction of dental pulp tissue from third molars of 15 healthy patients (age range: 20–35 years old) as described previously . All subjects signed the Ethical Committee consent brochure (Second University Internal Ethical Committee). Each subject underwent professional dental hygiene treatments for a week before tooth extraction. Only infection-free subjects were selected for cell collection. After mechanical and enzymatic digestion of the tissue with a collagenase I/dispase solution, the sample was filtered with 70 μm Falcon strainers (BD Pharmingen, Buccinasco, Milano, Italy) and centrifuged for 7 min at 1300 rpm. The pellets were then plated in T-25 flasks at 37 °C and 5% CO 2 in DMEM culture medium supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, and 100 U/ml penicillin and 100 mg/ml streptomycin (all purchased from GIBCO-Life Technologies, Monza, Italy). Adhered cells were expanded until they reached about 5 × 10 5 cells/flask.
2.3
FACS analysis and sorting
Cells were detached using trypsin–EDTA (GIBCO). At least 200,000 cells were incubated with fluorescent-conjugated antibodies for 30 min at 4 °C, washed, and re-suspended in PBS. The antibodies used in this study were: anti-CD34 PE (BD Pharmingen, Buccinasco, Milano, Italy) and anti-CD90 FITC (BD Pharmingen, Buccinasco, Milano, Italy). Isotypes were used as controls. Cells were analyzed with an Accuri C6 (BD Biosciences, San Jose, CA, USA) and data collected with FSC Express version 3 (De Novo Software). Cells were sorted using simultaneous positivity for CD90 and CD34 using a FACS ARIA III (BD, Franklin Lakes, NJ, USA). The purity of sorted populations was routinely 90%.
2.4
3D cell culture: In vitro tissue engineering
After proliferation, collected subpopulations were seeded – at a density of 5 × 10 5 cells/implant – onto dental implants that had been previously washed in PBS. After 1 h of incubation in 100 μl of culture medium to allow cell attachment, the cell–implant devices were transferred to 15 ml tubes with a cap filter and incubated with osteogenic medium in a humidified atmosphere at 37 °C and 5% CO 2 in a rotating culture apparatus (Wheaton Science Products, Millville, NJ, USA) at 6 rpm; cells plated in flasks were used as control. The 3D culture was performed for 21 days in osteogenic medium changed twice weekly; specimens were collected every seven days.
2.5
Cytotoxicity test on conditioned medium
Cytotoxicity was evaluated on cells cultured in medium conditioned by the presence of implants. The conditioned medium was prepared by incubating each implant in 3 ml of DMEM without phenol red and supplemented with antibiotics (penicillin, streptomycin), glutamine, and FBS at 37 °C for 3 days. DPSCs were plated in multiwells, cultured in conditioned medium for 24 h and 48 h, and cell viability determined by MTT colorimetric assay. The values are expressed as the percentage of cell viability compared with control (cells incubated in unconditioned culture medium). The measurements were performed in triplicate.
2.6
Proliferation assays
The MTT colorimetric assay was also performed to assess cell adhesion and proliferation. To this end, 5 × 10 5 cells were plated on implants and incubated, as described above, in DMEM supplemented with FBS, l -glutamine, and antibiotics. Seeded implants were collected after 24 h and 48 h of 3D culture: medium was removed and cell–implants incubated for 4 h in a solution of 5 mg/ml MTT. The same number of cells cultured in 2D was used as control. After medium removal, 300 μl of DMSO was added to each well containing seeded implants or control cells for 10 min; supernatants collected were read at 540 nm with a spectrophotometer. Cell viability was calculated proportionally to the quantity of formazan salts produced by the enzymatic activity of cells. Values are given as percentage versus the control, and normalized with respect to the number of cells and samples volume.
2.7
Immunofluorescence
Cell adhesion was evaluated by labeling with Hoechst 33342, a DNA-binding probe. After 3 and 5 days of culture, implants seeded with 1 × 10 6 cells/ml were washed in PBS and fixed with 4% paraformaldehyde (PFA) solution. Specimens were incubated in a 1:200 solution of 10 mg/ml Hoechst (Invitrogen) in PBS for 10 min in the dark. Images were collected under a fluorescence microscope (Axiovert 100; Zeiss).
2.8
Scanning electron microscopy
Adhered cell morphology was assessed by SEM (Supra 40 ZEISS, Weimar, Germany). Seeded implants were deprived of medium, washed, fixed in PFA, and post-fixed with 0.1% OsO 4 for 1 h. Thereafter, specimens were gradually dehydrated in an increasing ethanol concentration, treated by critical point drying, dry mounted on a stub, and sputter-coated with gold/palladium.
2.9
Histological evaluation
After 14 days of 3D culture, cell–implant biocomplexes were fixed in a solution of 4% PFA, dehydrated in an increasing gradient of alcohol, and embedded in glycolmethacrylate resin (Techonovit 7200 VLC; Kulzer, Wehrheim, Germany). Then, they were thin-sectioned (150 μm) with a Precise 1 Automated System (Assing, Rome, Italy) and reduced to about 30 μm with a specially designed grinding machine. The slides were stained with toluidine blue and acid fucsin, and images collected under transmitted light with a Leitz–Laborlux microscope (Laborlux S, Leitz, Wetzlar, Germany).
2.10
qRT-PCR
The osteoinduction capability of implants was evaluated by qRT-PCR analysis for genes involved in osteogenic differentiation on specimens collected after 7, 14, and 21 days of 3D cell culture. In particular, we examined the expression of genes involved in the production of molecules responsible for deposition of mineralized matrix: bone alkaline phosphatase ( BAP ), osteopontin ( OPN ), and osteocalcin ( OC ). RNA extracted from pellets of cells cultured in 2D was used as control. RNA from cells adhered on implants was extracted by processing the entire sample according to the protocol of the Ambion RNA extraction kit (Life Technologies). cDNA was obtained after treatment with DNase (Promega, Italy) and reverse transcriptase (ImProm–II Reverse Transcriptase). Quantitative Real-Time PCR was performed using the SYBR Green method and products were detected using a StepOne Real-Time PCR instrument (Applied Biosystems, California, USA). The amount of cDNA of the gene of interest was normalized to GAPDH cDNA. The following primers were used: BAP fw 5′-tcaaaccgagatacaagcac-3′, rev 5′-ggccagacgaaagatagagt-3′; OPN fw 5′-gccgaggtgatagtgtggtt-3′, rev 5′-tgaggtgatgtcctcgtctg-3′; OC fw 5′-ccctcacactcctcgccctatt-3′, rev 5′-aagccgatgtggtcagccaactcgt-3′.
2.11
ELISA for h-osteocalcin, h-VEGF
In order to evaluate levels of human OC and VEGF produced by the cells and released into the culture medium, supernatant was collected from 3D cultures after 7, 14, and 21 days of culture. After centrifugation to remove particulates, 2 ml aliquots of medium were stored at −20 °C until processing for analysis. The evaluation was carried out with an ELISA kit (Human Osteocalcin ELISA kit, Invitrogen; Human VEGF ELISA kit, Invitrogen) and concentrations read versus a standard curve at 450 nm using a spectrophotometer (DAS Plate Reader, Rome, Italy). The assays were performed in triplicate.
2.12
Statistical analysis
All experiments were carried out in triplicates. Data are expressed as mean ± standard deviation (SD). The level of significance was set at p ≤ 0.05. Statistical significance of the data was determined by t -test using Sigma Plot software.
2
Materials and methods
2.1
Dental implants
The dental implants used in this study were Tri-Vent, purchased from TRI Dental Implants Int AG (Baar, Switzerland), and TiUnite, purchased from Nobel Biocare (Nobel Biocare Goteborg, Sweden). Tri-Vent is a sandblasted, acid-etched surface created by blasting the implant surface under pressure; TiUnite is composed of a slightly rough titanium oxide layer covered with a phosphorus coat conferring it a ceramic-like property rich in micropores.
2.2
Cell extraction and 2D culture
Mesenchymal stem cells were obtained by the extraction of dental pulp tissue from third molars of 15 healthy patients (age range: 20–35 years old) as described previously . All subjects signed the Ethical Committee consent brochure (Second University Internal Ethical Committee). Each subject underwent professional dental hygiene treatments for a week before tooth extraction. Only infection-free subjects were selected for cell collection. After mechanical and enzymatic digestion of the tissue with a collagenase I/dispase solution, the sample was filtered with 70 μm Falcon strainers (BD Pharmingen, Buccinasco, Milano, Italy) and centrifuged for 7 min at 1300 rpm. The pellets were then plated in T-25 flasks at 37 °C and 5% CO 2 in DMEM culture medium supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, and 100 U/ml penicillin and 100 mg/ml streptomycin (all purchased from GIBCO-Life Technologies, Monza, Italy). Adhered cells were expanded until they reached about 5 × 10 5 cells/flask.
2.3
FACS analysis and sorting
Cells were detached using trypsin–EDTA (GIBCO). At least 200,000 cells were incubated with fluorescent-conjugated antibodies for 30 min at 4 °C, washed, and re-suspended in PBS. The antibodies used in this study were: anti-CD34 PE (BD Pharmingen, Buccinasco, Milano, Italy) and anti-CD90 FITC (BD Pharmingen, Buccinasco, Milano, Italy). Isotypes were used as controls. Cells were analyzed with an Accuri C6 (BD Biosciences, San Jose, CA, USA) and data collected with FSC Express version 3 (De Novo Software). Cells were sorted using simultaneous positivity for CD90 and CD34 using a FACS ARIA III (BD, Franklin Lakes, NJ, USA). The purity of sorted populations was routinely 90%.
2.4
3D cell culture: In vitro tissue engineering
After proliferation, collected subpopulations were seeded – at a density of 5 × 10 5 cells/implant – onto dental implants that had been previously washed in PBS. After 1 h of incubation in 100 μl of culture medium to allow cell attachment, the cell–implant devices were transferred to 15 ml tubes with a cap filter and incubated with osteogenic medium in a humidified atmosphere at 37 °C and 5% CO 2 in a rotating culture apparatus (Wheaton Science Products, Millville, NJ, USA) at 6 rpm; cells plated in flasks were used as control. The 3D culture was performed for 21 days in osteogenic medium changed twice weekly; specimens were collected every seven days.
2.5
Cytotoxicity test on conditioned medium
Cytotoxicity was evaluated on cells cultured in medium conditioned by the presence of implants. The conditioned medium was prepared by incubating each implant in 3 ml of DMEM without phenol red and supplemented with antibiotics (penicillin, streptomycin), glutamine, and FBS at 37 °C for 3 days. DPSCs were plated in multiwells, cultured in conditioned medium for 24 h and 48 h, and cell viability determined by MTT colorimetric assay. The values are expressed as the percentage of cell viability compared with control (cells incubated in unconditioned culture medium). The measurements were performed in triplicate.
2.6
Proliferation assays
The MTT colorimetric assay was also performed to assess cell adhesion and proliferation. To this end, 5 × 10 5 cells were plated on implants and incubated, as described above, in DMEM supplemented with FBS, l -glutamine, and antibiotics. Seeded implants were collected after 24 h and 48 h of 3D culture: medium was removed and cell–implants incubated for 4 h in a solution of 5 mg/ml MTT. The same number of cells cultured in 2D was used as control. After medium removal, 300 μl of DMSO was added to each well containing seeded implants or control cells for 10 min; supernatants collected were read at 540 nm with a spectrophotometer. Cell viability was calculated proportionally to the quantity of formazan salts produced by the enzymatic activity of cells. Values are given as percentage versus the control, and normalized with respect to the number of cells and samples volume.
2.7
Immunofluorescence
Cell adhesion was evaluated by labeling with Hoechst 33342, a DNA-binding probe. After 3 and 5 days of culture, implants seeded with 1 × 10 6 cells/ml were washed in PBS and fixed with 4% paraformaldehyde (PFA) solution. Specimens were incubated in a 1:200 solution of 10 mg/ml Hoechst (Invitrogen) in PBS for 10 min in the dark. Images were collected under a fluorescence microscope (Axiovert 100; Zeiss).
2.8
Scanning electron microscopy
Adhered cell morphology was assessed by SEM (Supra 40 ZEISS, Weimar, Germany). Seeded implants were deprived of medium, washed, fixed in PFA, and post-fixed with 0.1% OsO 4 for 1 h. Thereafter, specimens were gradually dehydrated in an increasing ethanol concentration, treated by critical point drying, dry mounted on a stub, and sputter-coated with gold/palladium.
2.9
Histological evaluation
After 14 days of 3D culture, cell–implant biocomplexes were fixed in a solution of 4% PFA, dehydrated in an increasing gradient of alcohol, and embedded in glycolmethacrylate resin (Techonovit 7200 VLC; Kulzer, Wehrheim, Germany). Then, they were thin-sectioned (150 μm) with a Precise 1 Automated System (Assing, Rome, Italy) and reduced to about 30 μm with a specially designed grinding machine. The slides were stained with toluidine blue and acid fucsin, and images collected under transmitted light with a Leitz–Laborlux microscope (Laborlux S, Leitz, Wetzlar, Germany).
2.10
qRT-PCR
The osteoinduction capability of implants was evaluated by qRT-PCR analysis for genes involved in osteogenic differentiation on specimens collected after 7, 14, and 21 days of 3D cell culture. In particular, we examined the expression of genes involved in the production of molecules responsible for deposition of mineralized matrix: bone alkaline phosphatase ( BAP ), osteopontin ( OPN ), and osteocalcin ( OC ). RNA extracted from pellets of cells cultured in 2D was used as control. RNA from cells adhered on implants was extracted by processing the entire sample according to the protocol of the Ambion RNA extraction kit (Life Technologies). cDNA was obtained after treatment with DNase (Promega, Italy) and reverse transcriptase (ImProm–II Reverse Transcriptase). Quantitative Real-Time PCR was performed using the SYBR Green method and products were detected using a StepOne Real-Time PCR instrument (Applied Biosystems, California, USA). The amount of cDNA of the gene of interest was normalized to GAPDH cDNA. The following primers were used: BAP fw 5′-tcaaaccgagatacaagcac-3′, rev 5′-ggccagacgaaagatagagt-3′; OPN fw 5′-gccgaggtgatagtgtggtt-3′, rev 5′-tgaggtgatgtcctcgtctg-3′; OC fw 5′-ccctcacactcctcgccctatt-3′, rev 5′-aagccgatgtggtcagccaactcgt-3′.
2.11
ELISA for h-osteocalcin, h-VEGF
In order to evaluate levels of human OC and VEGF produced by the cells and released into the culture medium, supernatant was collected from 3D cultures after 7, 14, and 21 days of culture. After centrifugation to remove particulates, 2 ml aliquots of medium were stored at −20 °C until processing for analysis. The evaluation was carried out with an ELISA kit (Human Osteocalcin ELISA kit, Invitrogen; Human VEGF ELISA kit, Invitrogen) and concentrations read versus a standard curve at 450 nm using a spectrophotometer (DAS Plate Reader, Rome, Italy). The assays were performed in triplicate.
2.12
Statistical analysis
All experiments were carried out in triplicates. Data are expressed as mean ± standard deviation (SD). The level of significance was set at p ≤ 0.05. Statistical significance of the data was determined by t -test using Sigma Plot software.
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