Volume 10 | Issue 9 | September 2017
Release of VEGF from Dental Implant Improves Osteogenetic Process: Preliminary In Vitro Tests
2.1. In Vitro Release of VEGF
In order to test the ability of VEGF-enriched dental implants to release VEGF in vitro, we quantified the growth factor in the cell culture medium up to 7 days by means of an ELISA test. As reported in Figure 1, VEGF concentration increases in the medium in a time-dependent manner, starting from a basal concentration of 180 ng/mL after 1 h and reaching the maximum level of 1800 ng/mL after 24 h.
Figure 1. Quantification of Vascular Endothelial Growth Factor (VEGF) release in the cell culture medium. VEGF concentration increases in the medium in a time-dependent manner, reaching a plateau after 24 h.
2.2. Biocompatibility and Cell Proliferation
Figure 2. (a,b) 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. (a: in form of bars and b: in form of line). Cells are able to proliferate on both the control and VEGF-enriched surface from 3 to 28 days of culture with no statistical difference between the two surfaces. On the contrary, when cells are treated with Tumor Necrosis Factor α (TNFα) their proliferation is significantly reduced on both the control and the VEGF-enriched implant; (c,d) Quantification of intracellular Lactate Dehydrogenase (LDH) activity (c: in form of bars; d: in form of lines). Intracellular LDH activity proves that cells are able to produce metabolites if seeded onto both surfaces, with improved results after 7 days from seeding; (e,f) Quantification of extracellular LDH activity (e: in form of bars; f: in form of lines). Extracellular LDH activity confirms that metabolites are secreted by the cells and are not associated with damage of the membrane. Statistically significant differences are indicated as * p < 0.05, ** p < 0.01, and compared with the control condition.
2.3. Intracellular and Extracellular Lactate Dehydrogenase (LDH) Activity
2.4. Reactive Oxygen Species (ROS) Production
Figure 3. (a,b) (a: in form of bars and b: in form of line). Reactive Oxygen Species (ROS) production of Dental Pulp Stem Cells (DPSCs) seeded onto VEGF-enriched or control dental implants. Histograms show a slight time-dependent increase in metabolic activity in cells seeded onto both surfaces. The presence of VEGF on the implant surface is able to reduce the intracellular ROS production, especially under inflammatory conditions. Results are expressed as fluorescent arbitrary units per second (f.a.u./s); (c,d) (c: in form of bars and d: in form of line). Evaluation of Senescence-associated beta-galactosidase (SA-b GAL) (SA-b GAL) activity of DPSCs under normal or inflammatory conditions on surfaces combined or not with VEGF. Results show that cells cultured on implants loaded with VEGF have a lower SA-b GAL activity value compared to the cells seeded on the control one.
2.6. Cell Morphology
Figure 4. Immunofluorescence staining of the actin filament with phalloidin (in red). Cell nuclei are counterstained with Hoechst (in blue). (a for control; b on VEGF implants) After 7 days of culture, DPSCs have colonized the implant surface showing a star-like morphology; (c for control; d on VEGF implants) Cells are completely spread onto the implant surface after 14 days from seeding.
A detailed analysis of cell shape has additionally been performed. In particular, the Circularity, Roundness, and Solidity parameters have been considered. As shown in Figure 5, when cells were seeded onto the VEGF-enriched implants and cultured for 14 days, they assumed an elongated morphology, as suggested by the Circularity and Roundness calculated values. This would confirm that the cells lost the staminal rounded aspect for acquiring a more elongated morphology.
Figure 5. Analysis of the (a) Circularity; (b) Roundness; and (c) Solidity cell shape parameters after 7 and 14 days of culture onto the control and VEGF-enriched implants. * p < 0.05.
2.7. Effect of VEGF on Endothelial Cell Differentiation
Figure 6. Immunofluorescence staining of the CD31 endothelial marker in DPSCs seeded onto (a) control implant; (b) implant loaded with VEGF; (c) control implant under inflammatory conditions; (d) VEGF-enriched implant under inflammatory conditions; (e) Evaluation of the % of CD31-positive cells.
2.8. Osteogenic Commitment
Figure 7. Alkaline phospahatase (ALP) production profiles in DPSCs seeded onto polystyrene, control or VEGF-enriched implants under normal or inflammatory conditions.
4. Materials and Methods
4.1. Dental Implants
Dental implants were supplied by Ditron Dental (Ashkelon, Israel). In particular, MPI—Molecular Precision Implants with dimensions of 8 mm × 4.2 mm enriched with VEGF were used for the in vitro tests (Figure 9). The same implants without VEGF were used as control implants.
4.2. Quantitative Analysis of VEGF Release
4.3. DPSCs Isolation and Seeding onto Dental Implant
4.4. MTT Assay
4.5. LDH Activity
4.6. ROS Measurements
4.7. SA-b GAL Staining
4.8. Cell Shape Analysis
Cell shape was evaluated on samples stained for 40 min with 5 mg/mL phalloidin. Briefly, cells were fixed in 4% paraformaldehyde in PBS for 10 min, then permeabilized with 0.1% triton X-100 (Sigma-Aldrich, Saint Louis, MA, USA) in PBS for 30 min at room temperature. Phalloidin was then used for fluorescent staining of actin filaments, whilst nuclear staining was performed with 2 μg/mL Hoechst H33342 (Sigma-Aldrich) solution for 5 min. Images were acquired with the inverted optical microscope DMI4000 B (Leica Microsystems, Wetzlar, Germany). Then, ImageJ software (http://rsb.info.nih.gov/ij) was used to calculate cell area and different shape descriptors of at least 30 distinct cells. In detail, the Circularity (C), Roundness (R), and Solidity (S) of cells were calculated according to the following equations :
Circularity = C = 4πA/P2
where A is the cell area and P is the perimeter;
Roundness = R = a/b
where a and b are the width and length of the minimum bounding, respectively;
Solidity = S = A/ConvexA
where ConvexA is the area enclosed by the smallest shell that borders all the points of the cell.
4.10. Real-Time RT-PCR
4.11. Statistical Analyses
4.12. ALP Activity Measurements
Thereafter, 50 μL of 5 mM pNPP solution was added to each well containing test samples and background control and incubated for 60 min at 25 °C, protecting the plate from the light. A standard curve of 0, 4, 6, 12, 16 and 20 nmol/well was generated from 1 mM pNPP standard solution bringing the final volume to 120 μL with Assay Buffer. All reactions were then stopped by adding 20 μL of Stop solution into each standard and sample reaction except the sample background control reaction. Optical density was read at 405 nm in a microplate reader (Victor). The results were normalized subtracting the value derived from the zero standards from all standards, samples and sample background control. The pNP standard curve was plotted to identify the pNP concentration in each sample. ALP activity of the test samples was calculated as follow:
where A is the amount of pNP generated by samples (in μmol). V is the amount of sample added in the assay well (in mL). T is the reaction times (in minutes).
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