3D printing of strontium-enriched biphasic calcium phosphate scaffolds for bone regeneration.

Calcium phosphate (CaP) scaffolds doping with therapeutic ions are one of the focuses of recent bone tissue engineering research. Among the therapeutic ions, strontium stands out for its role in bone remodeling. This work reports a simple method to produce Sr-doped 3D-printed CaP scaffolds, using Sr-doping to induce partial phase transformation from ß-tricalcium phosphate (ß-TCP) to hydroxyapatite (HA), resulting in a doped biphasic calcium phosphate (BCP) scaffold. Strontium carbonate (SrCO3) was incorporated in the formulation of the 3D-printing ink, studying ß-TCP:SrO mass ratios of 100:0, 95:5, and 90:10 (named as ß-TCP, ß-TCP/5-Sr, and ß-TCP/10-Sr, respectively). Adding SrCO3 in the 3D-printing ink led to a slight increase in viscosity but did not affect its printability, resulting in scaffolds with a high printing fidelity compared to the computational design. Interestingly, Sr was incorporated into the lattice structure of the scaffolds, forming hydroxyapatite (HA). No residual SrO or SrCO3 were observed in the XRD patterns of any composition, and HA was the majority phase of the ß-TCP/10-Sr scaffolds. The addition of Sr increased the compression strength of the scaffolds, with both ß-TCP/5-Sr and ß-TCP/10-Sr performing better than the ß-TCP. Overall, ß-TCP/5-Sr presented higher mineralized nodules and mechanical strength, while ß-TCP scaffolds presented superior cell viability. The incorporation of SrCO3 in the ink formulation is a viable method to obtain Sr-BCP scaffolds. Thus, this approach could be explored with other CaP scaffolds aiming to optimize their performance and the addition of alternative therapeutic ions.

Influence of titanium and zirconia substrates on the synthesis of inflammatory mediators.

The repair and homeostasis of peri-implant tissues depend on several factors such as the local presence of pathogenic bacteria and their products. Among other events, peri-implant tissue response is also related to the implant material used, which interferes with cells and extracellular matrix interactions, affecting the osseointegration process. In this study, the influence of zirconia (Zr) and titanium (Ti) substrates on the response of preosteoblasts (MC3T3) and murine macrophages (RAW 264.7) exposed to lipopolysaccharide (LPS, P. gingivalis) was evaluated. Zr and Ti disks were obtained and subjected to surface roughness standardization, which was analyzed by scanning electronic microscopy (SEM). The cells were subsequently cultured on Zr and Ti surfaces in AlphaMEM culture medium for 24 h, followed by LPS stimulus for 4 h. The production of reactive oxygen species (ROS) and gene expression of inflammatory markers were determined. SEM images showed that Ti disks exhibited higher surface roughness than that of Zr disks. Cells that seeded onto Ti and Zr had increased expression of inflammatory mediators and ROS production in the presence of LPS; however, such cell responses were more evident for Ti disks. These data indicate that contact of cells with Zr surfaces may lead to a lower inflammatory potential than Ti surfaces. Elucidation of the inflammatory response triggered by LPS for cells in contact with titanium and zirconia may contribute to the selection of materials for installation of osseointegrated implants.

EGF coating of titanium surfaces modulates cytokines in oral mucosal primary cells exposed to TNF-α.

OBJECTIVE: This study assessed the metabolism of oral mucosal cells cultured on titanium discs (Ti) coated (or not) with epidermal growth factor (EGF) and exposed to tumor necrosis factor alpha (TNF-α). METHODS: Fibroblasts or keratinocytes were seeded on Ti coated or not with EGF, and then exposed to 100 ng/mL of TNF-α for 24 h. Groups were established: G1: Ti (control); G2: Ti + TNF-α; G3: Ti + EGF; and G4: Ti + EGF + TNF-α. Both cell lines were evaluated for: viability (AlamarBlue®, n = 8); interleukin 6 and 8 (IL-6, IL-8) gene expression (qPCR, n = 5), and protein synthesis (ELISA, n = 6). For keratinocytes cells, the matrix metalloproteinase type 3 (MMP-3) was evaluated by qPCR (n = 5) and ELISA (n = 6). A 3-D culture of fibroblasts was analyzed by confocal microscopy. The data were subjected to ANOVA analysis, α = 5%. RESULTS: Increased cell viability was observed in all groups compared with G1. Enhanced gene expression and synthesis of IL-6 and IL-8 by fibroblasts and keratinocytes in G2 and modulation of hIL-6 gene expression in G4 was noted. Modulation of IL-8 synthesis occurred in keratinocytes in G3 and G4. Keratinocytes in G2 showed enhanced gene expression of hMMP-3. A 3-D culture showed more cells in G3. Fibroblasts in G2 exhibited disrupted cytoplasmic membrane. Cells in G4 showed elongated morphology with intact cytoplasm. CONCLUSIONS: EGF coating increases cell viability and modulates the response of oral cells exposed to an inflammatory stimulus.

Naringenin and proanthocyanidins pre-treatment decreases synthesis and activity of gelatinases induced by zoledronic acid in a dental implant surface in vitro model.

OBJECTIVE: To assess the effects of pre-treatment with proanthocyanidins (PA) flavonoids, from grape seed extract, and synthetic naringenin (NA) on the synthesis of matrix metalloproteinases (MMPs) gelatinases and their tissue inhibitors (TIMPs), as well as the gelatinolytic activity of MMPs by human gingival fibroblasts (HGF) and osteoblasts (Ob) exposed to zoledronic acid (ZA) in a dental implant surface in vitro model. DESIGN: The highest non-cytotoxic concentrations of NA and PA were determined for HGF (10 µg/mL; defined by previous study) and Ob (0.5 µg/mL; defined by prestoBlue assay). Then, HFG and Ob were individually seeded onto titanium discs, and after 24 h, cells were pre-treated (or not) with NA or PA, followed (or not) by exposure to ZA. Next, MMP-2, MMP-9, TIMP-1, TIMP-2 synthesis (ELISA), and gelatinolytic activity (in situ zymography) was evaluated. Data were analyzed by one-way ANOVA and Tukey tests (α = 0.05). RESULTS: ZA treatment increased the synthesis (p < 0.05) and activity of MMPs; flavonoids pre-treatment controlled ZA-induced gelatinolytic effects, down-regulating MMPs synthesis (p < 0.05) and activity by HGF and Ob. For HGF, NA and PA pre-treatment did not up-regulate TIMP synthesis after ZA exposure (p > 0.05); for Ob, TIMP-2 was up-regulated (p < 0.05) by flavonoids, followed by ZA. CONCLUSIONS: NA and PA pre-treatment provides interesting results in the modulation of ZA deleterious effects, down-regulating MMP-2 and MMP-9 synthesis and activity by HGF and Ob and up-regulating TIMP-2 by Ob.

Regulation of interleukin-6 and matrix metalloproteinases syntheses by bioflavonoids and photobiomodulation in human gingival fibroblasts.

This study aimed to evaluate the separately effects of bioflavonoids proanthocyanidins, from grape seed extract (GSE) and synthetic naringenin (NA), as well as photobiomodulation (PBM) by low-level laser therapy on interleukin (IL)-6 and matrix metalloproteinases (MMPs) syntheses by human gingival fibroblasts (HGF). For this purpose, a connective tissue exposure (ulceration) model of HGF, stimulated with tumor necrosis factor-alpha (TNF-α), was used. Initially, the highest non-cytotoxic and non-genotoxic concentrations of bioflavonoids were determined by cell viability and micronuclei formation assays. Then, HGF were exposed to different stimuli: culture medium (negative control), dimethyl sulfoxide (DMSO), TNF-α, NA, GSE, TNF-α + NA, TNF-α + GSE, PBM (3 J/cm2, 0.025 W, 780 nm), and TNF-α + PBM. Next, IL-6, MMP-2, and MMP-9 syntheses were assessed. The concentration of 10 µg/mL of bioflavonoids increased cell viability at 24 and 48 h and did not present cytotoxic or genotoxic effects on HGF after 24, 48, and 72 h of contact. This concentration was selected for the assessment of bioflavonoids potential in modulating inflammatory mediators. TNF-α exposure enhanced IL-6 (170%), MMP-2 (10%), and MMP-9 (20%) syntheses, while a decrease of MMP-2 by 55% after exposure to TNF-α + GSE and 20% after TNF-α + NA and TNF-α + PBM was observed. MMP-9 synthesis was decreased by 35% after TNF-α + NA, 20% after TNF-α + GSE, and 30% after PBM. IL-6 was down-regulated by GSE in the presence of TNF-α (80%). In conclusion, TNF-α up-regulated IL-6 and MMPs, while bioflavonoids and PBM down-regulated MMP-2 and MMP-9 syntheses; GSE also decreased IL-6 synthesis, demonstrating the individual promising potential of these therapies for ulceration management.

Titanium alkalinization improves response of osteoblasts to zoledronic acid.

This investigation is aimed to determine the effect of the modification of titanium surface with NaOH on the metabolism of osteoblasts treated with zoledronic acid (ZA). Machined and NaOH-treated titanium disks were used. Surfaces were characterized by scanning electron microscopy, confocal microscopy, and x-ray photoelectron spectroscopy (XPS) analysis. Human osteoblasts were seeded onto the disks. After 24 h, cells were treated with ZA at 5 µM for 7 days. At this point, cell viability, collagen synthesis, total protein production, alkaline phosphatase activity, and mineral nodule deposition were assessed. The results of surface roughness were descriptively and statistically analyzed (t-Student), while the XPS results were qualitatively described. Cell metabolism data were analyzed by the analysis of variance two-way and Tukey tests at a 5% significance level. The results demonstrated that NaOH-treatment increased surface roughness (p < .05) and confirmed the presence of sodium titanate and a pH switch on the NaOH-treated disks. This modification also resulted in higher cell viability, collagen synthesis, total protein production, and alkaline phosphatase by osteoblasts when compared to cells seeded onto machined disks (p < 0.05). In the presence of ZA, all cellular metabolism and differentiation parameters were significantly reduced for cells seeded on both surfaces (p < 0.05); however, the cells seeded onto modified surfaces showed higher values for these parameters, except for mineral nodule deposition (p < 0.05). NaOH modification improved cell adhesion and metabolism of osteogenic cells even in the presence of ZA. The surface modification of titanium with NaOH solution may be an interesting strategy to improve metabolism and differentiation of osteoblasts and accelerate osseointegration process, mainly for tissues exposed to ZA.

Photobiomodulation using LLLT and LED of cells involved in osseointegration and peri-implant soft tissue healing.

This study evaluated the influence of photobiomodulation (PBM) using low-level laser therapy (PBM/LLLT) or light-emitting diode (PBM/LED) therapy on peri-implant tissue healing. A laboratory model was used to assess the adhesion and metabolism of osteoblasts (SaOs-2), human gingival fibroblasts (HGF), and normal oral keratinocytes (NOK) seeded on a titanium (Ti) surface. After seeding the cells on disks of Ti placed in wells of 24-well plates, three irradiations were performed every 24 h at energy density of 3 J/cm2. For PBM/LLLT, a LaserTABLE device was used with a wavelength of 780 nm and 25 mW, while for PBM/LED irradiation, a LEDTABLE device was used at 810 nm, 20 mW, at a density of 3 J/cm2. After irradiations, the number of cells (NC) attached and spread on the Ti surface, cell viability (CV), total protein (TP), and collagen (Col) synthesis were assessed. Alkaline phosphate activity (ALP) was evaluated only for SaOs-2. Data were submitted to ANOVA complemented by Turkey statistical tests at a 5% significance level. PBM significantly increased adherence of NOK to the Ti surface, while no significant effect was observed for SaOs-2 and HGF. PBM positively affected CV, as well as Col and TP synthesis, in distinct patterns according to the cell line. Increased ALP activity was observed only in those cells exposed to PBM/LLLT. Considering cell specificity, this investigation reports that photobiomodulation with low-power laser and LED at determined parameters enhances cellular functions related to peri-implant tissue healing in a laboratory model.

Influence of bisphosphonates on oral implantology: Sodium alendronate and zoledronic acid enhance the synthesis and activity of matrix metalloproteinases by gingival fibroblasts seeded on titanium.

OBJECTIVE: This study aimed to assess the influence of the bisphosphonates zoledronic acid and sodium alendronate on MMP-2 and MMP-9 synthesis and activity by gingival fibroblasts seeded onto titanium substrate. DESIGN: Titanium discs were placed in 24-well cell culture plates and gingival fibroblasts were seeded (1 × 105 cells/discs) on them using Dulbecco's Modified Eagle's Medium (DMEM) + 10 % fetal bovine serum (FBS) for 24 h. After this period, a fresh serum-free DMEM containing zoledronic acid or sodium alendronate at 0.5 µM, 1 µM or 5 µM was applied on the cells for an additional of 24 h. Serum-free DMEM and tumor necrosis factor alpha (TNF-α) were used as negative and positive controls, respectively. MMP-2 and MMP-9 synthesis and activity were determined by ELISA (Enzyme-Linked Immunosorbent Assay) and conventional/in situ zymography. Quantitative data were analyzed by one-way ANOVA and Tukey's tests (α = 0.05). The in situ zymography data were qualitatively described. RESULTS: Despite both bisphosphonates increased the MMPs synthesis, this effect was significant higher in zoledronic acid groups. MMPs activity resembled by gelatinolytic activity was also enhanced by sodium alendronate and zoledronic acid in a similar pattern. CONCLUSIONS: Zoledronic acid and sodium alendronate increased in a dose-dependent manner MMP-2 and MMP-9 synthesis by gingival fibroblasts seeded on titanium. MMP-2 activity was up-regulated by zoledronic acid treatment.

Chemotherapy drugs and inflammatory cytokines enhance matrix metalloproteinases expression by oral mucosa cells.

OBJECTIVE: Oral mucositis (OM), the most common side effect of cancer therapy, is associated with pro-inflammatory cytokines and matrix metalloproteinases (MMPs) increased expression. Although there are approaches for OM management, none is infallible, thus, elucidation of molecular events related to OM etiopathogenesis may improve current therapeutic strategies. This study assessed the influence of pro-inflammatory cytokines and chemotherapy drugs on MMPs expression and synthesis by oral mucosa cells. DESIGN: Human gingival fibroblasts (HGF) were exposed to different concentrations of methotrexate (MTX) and 5-fluorouracil (5-FU); subsequentially, cell viability, nitric oxide and interleukin(IL)-6 production were evaluated to select the concentration of these drugs that could stimulate inflammatory phenotype without cytotoxic effects. Then, HGF and primary gingival keratinocytes (PGK) were subjected to different stimuli: culture medium (negative control), tumor necrosis factor-alpha (TNF-α - positive control), IL-6, IL-8, MTX, and 5-FU for 3, 6, 12, and 24 h. Next, gene expression and synthesis of MMP-2 and MMP-9 by HGF and MMP-3 by PGK were assessed. RESULTS: At 6 h, MMP-2 synthesis increased 60 % after exposure to TNF-α and MTX, 40 % after IL-6, and 15 % after IL-8. At 12 h, MMP-9 synthesis increased 15 % after exposure to TNF-α, while MMP-3 synthesis increased 30 % after TNF-α, and 10 % after IL-8. TNF-α-treated groups presented increased gene expression of all MMPs evaluated. IL-8 and 5-FU increased MMP-2 and MMP-3 expression, while IL-6 and MTX augmented MMP-2 expression. CONCLUSIONS: The chemotherapy drugs and cytokines investigated up-regulated MMPs expression by oral mucosa cells, which may lead to OM establishment and severity.

Effects of EGF-coated titanium surfaces on adhesion and metabolism of bisphosphonate-treated human keratinocytes and gingival fibroblasts.

OBJECTIVE: To assess the effects of epidermal growth factor (EGF)-coated titanium (Ti) discs on the adhesion and metabolism of keratinocytes and gingival fibroblasts exposed to nitrogen-containing bisphosphonates. MATERIALS AND METHODS: Keratinocytes and fibroblasts were seeded (1 × 105 cells/disc) on Ti discs coated with EGF (100 nM). After 24 h, cells were exposed or not to sodium alendronate (SA) or zoledronic acid (ZA) at different concentrations (0 = control, 0.5, 1, or 5 µM) for 48 h. Cell adhesion to the substrates was evaluated by fluorescence microscopy. Cell viability (alamarBlue, n = 6) and synthesis of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and keratinocytes growth factor (KGF) (ELISA, n = 6) were assessed. Data were statistically analyzed by one-way ANOVA and Tukey tests (α = 0.05). RESULTS: Higher cell adhesion rate was observed when keratinocytes and fibroblasts were seeded onto EGF-coated discs in comparison to uncoated discs. ZA treatment hindered the adhesion of both cell lines on the Ti discs as well as reduced the viability and synthesis of VEGF, KGF and MMP-2 by cells (p < 0.05). SA treatment did not affect cell viability, but interfered negatively on the adhesion and synthesis of EGF and KGF by the cells (p < 0.05). EGF-coated surface increased cell viability and synthesis of growth factors as well as downregulated the synthesis of MMP-2 in comparison to control (p < 0.05). CONCLUSION: EGF applied on Ti surface improves the biological responses of oral mucosa cells exposed to SA and ZA. CLINICAL RELEVANCE: EGF-coating on titanium may be a suitable strategy to improve oral mucosa cellular events related to biological sealing, especially for patients under bisphosphonate therapy.

Photobiomodulation of inflammatory-cytokine-related effects in a 3-D culture model with gingival fibroblasts.

The aim of this study was to assess the effects of IL-6 and IL-8 cytokines on human gingival fibroblasts (HGF) cultured in a 3-D model and the possible photobiomodulation (PBM) of such effects by low-level laser therapy. In complete culture medium (DMEM), HGF from a healthy patient were seeded in a type I collagen matrix inserted into 24-well plates. After 5 days of incubation, the cytokines were added or not to serum-free DMEM, which was applied to the cell-enriched matrices. Then, PBM was performed: three consecutive irradiations using LaserTable diode device (780 nm, 0.025 W) at 0.5 J/cm2 were delivered or not to the cells. Twenty-four hours after the last irradiation, cell viability and morphology, gene expression, and synthesis of inflammatory cytokines and growth factors were assessed. The histological evaluation demonstrated that, for all groups, matrices presented homogeneous distribution of cells with elongated morphology. However, numerous cytokine-exposed cells were rounded. IL-6 and IL-8 decreased cell viability, synthesis of VEGF, and gene expression of collagen type I. PBM enhanced cell density in the matrices and stimulated VEGF expression, even after IL-6 challenge. Reduced TNF-α synthesis occurred in those cells subjected to PBM. In conclusion, PBM can penetrate collagen matrix and stimulate HGF, highlighting the relevance of this research model for further phototherapy studies and in vitro biomodulation of the healing process.

Epithelial cell-enhanced metabolism by low-level laser therapy and epidermal growth factor.

Reepithelialization and wound closure are the desired outcome for several ulcerative conditions. Such resolution reduces the possibility of wound contamination and maintenance of the injury and improves the reestablishment of tissue morphology and functions. Investigators are seeking adjuvant therapies that can accelerate wound healing and are developing new strategies for clinical applications. This study compared the effects of epidermal growth factor (EGF) application and low-level laser therapy (LLLT) on cultured epithelial cells. Cells were seeded in 24-well plates. After a 24-h incubation, the epithelial cells were either treated with EGF (100 µM in serum-free DMEM for 72 h) or subjected to LLLT (780 nm, 25 mW, 0.5, 1.5, and 3 J/cm2) by three applications every 24 h. Seventy-two hours after cells were treated with EGF or LLLT, cell migration, viability, proliferation, and collagen synthesis were assessed. Cells treated with EGF showed increased cell viability, proliferation, and collagen synthesis compared with those cells that received no treatment. LLLT enhanced cell migration; however, no significant effects of laser irradiation on other cell functions were observed. Comparison of both therapies demonstrated that EGF and LLLT enhanced specific epithelial cell activities related to wound healing.