Adipogenesis-Related Metabolic Condition Affects Shear-Stressed Endothelial Cells Activity Responding to Titanium

Abstract

Purpose: Obesity has increased around the world. Obese individuals need to be better assisted, with special attention given to dental and medical specialties. Among obesity-related complications, the osseointegration of dental implants has raised concerns. This mechanism depends on healthy angiogenesis surrounding the implanted devices. As an experimental analysis able to mimic this issue is currently lacking, we address this issue by proposing an in vitro high-adipogenesis model using differentiated adipocytes to further investigate their endocrine and synergic effect in endothelial cells responding to titanium.

Materials and methods: Firstly, adipocytes (3T3-L1 cell line) were differentiated under two experimental conditions: Ctrl (normal glucose concentration) and High-Glucose Medium (50 mM of glucose), which was validated using Oil Red O Staining and inflammatory markers gene expression by qPCR. Further, the adipocyte-conditioned medium was enriched by two types of titanium-related surfaces: Dual Acid-Etching (DAE) and Nano-Hydroxyapatite blasted surfaces (nHA) for up to 24 h. Finally, the endothelial cells (ECs) were exposed in those conditioned media under shear stress mimicking blood flow. Important genes related to angiogenesis were then evaluated by using RT-qPCR and Western blot.

Results: Firstly, the high-adipogenicity model using 3T3-L1 adipocytes was validated presenting an increase in the oxidative stress markers, concomitantly with an increase in intracellular fat droplets, pro-inflammatory-related gene expressions, and also the ECM remodeling, as well as modulating mitogen-activated protein kinases (MAPKs). Additionally, Src was evaluated by Western blot, and its modulation can be related to EC survival signaling.

Conclusion: Our study provides an experimental model of high adipogenesis in vitro by establishing a pro-inflammatory environment and intracellular fat droplets. Additionally, the efficacy of this model to evaluate the EC response to titanium-enriched mediums under adipogenicity-related metabolic conditions was analyzed, revealing significant interference with EC performance. Altogether, these data gather valuable findings on understanding the reasons for the higher percentage of implant failures in obese individuals.

Keywords: adipogenesis; angiogenesis; bone; dental implants; failure; obese; titanium; wound healing.

Figure 1

Outline and workflow. To evaluate the ECs behavior in response to titanium-enriched medium in concomitance with high adipogenesis conditions arising from 3T3-L1 adipocytes, we collected the medium conditioned by the adipocytes during their differentiation, which was later enriched with titanium for up to 24 h, as recommended by ISO 10993:2016. This conditioned medium was also further used to expose ECs for 72 h under shear stress mimicking blood flow, at which point the samples were collected to allow the molecular analysis.

Figure 2

High-glucose medium affects adipocyte differentiation. The 50 mM high-glucose medium was used to expose pre-adipocyte cells for stimulation to differentiation. Firstly, cytotoxicity was measured by using an MTT assay (A). Thereafter, oxidative stress was measured by evaluating the protein carbonylation (nmol/mg protein) by performing a method using DNPH (2,4-dinitrophenylhydrazine derivatizing agent) (B). The data are plotted respecting mean ± SD (n = 3), and the significance was shown using Student’s t-test, ** p = 0.0058. Intracellular fat droplets of the pre-adipocytes were acquired using a light microscope (40× magnification) thereafter stained by using Oil Red O Staining (C). HGM: high-glucose medium.

Figure 3

Adipogenesis model recapitulates the inflammation microenvironment and requires PPAR-γ. Pre-adipocytes were differentiated for 10 days using classical model when the cells were harvested and the biological samples forwarded to perform the qPCR technology. A significantly higher expression of IL-1β (A), IL-6 (B), IL-13 (C), IL-18 (D), IL-33 (E), TNF-α (G), and PPAR-γ (J) genes were observed in the adipocytes responding to HGM (50 mM). The graphs bring the n-fold change of the profile of gene expression normalized to the GAPDH gene (housekeeping gene). Significant differences were considered when * p < 0.05, and ** p < 0.01, *** p < 0.001. HGM: high glucose medium.

Figure 4

Adipocytes require MAPK and MMP activity. Adipocytes require MAPK-ERK (A,B), while the MAPK-P38 protein remains unchanged (C,D). β-Actin was used as the protein loading control. Additionally, higher activities of MMP9 (F–H) and MMP2 (I,J) were found in adipocytes responding to HGM. Data are plotted as means ± standard deviations (n = 3). Significant differences were considered when * p < 0.05, and ** p < 0.01. HGM: high glucose medium.

Figure 5

VEGF and VEGFR1 genes are modulated in response to titanium-based surfaces. Both genes are related to EC phenotype as well as to angiogenesis. Their response to H_Adip and the titanium-enriched medium seems to be relevant to the lower angiogenesis profile in adipogenesis. The VEGF gene presented a low expression profile in ECs responding to DAE, while there was no significance when considering the other groups (A). Additionally, the VEGFR1 gene presented a low profile of expression in ECs responding to nHA (B). The data show the n-fold changes in the profile of transcripts normalized to the 18 S gene (housekeeping gene). Differences were considered statistically significant when * p < 0.05, and *** p < 0.001, represented by red * when compared to the H_Adip group.

Figure 6

Both survival and cell proliferation progress were investigated in ECs. ECs require an increase in survival and cell cycle-related gene expression even more responding to high-adipogenesis condition, observing the behavior of AKT (A), CDK2 (B), and CDK4 (C). Differences were considered statistically when * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, represented by black * when compared to the Ctrl group, by red * when compared to the H_Adip group, and by green * when compared between the groups with nHA.

Figure 7

MAPK-related genes were modulated in EC responding to adipogenesis. qPCR shows different modulations of the MAPKs genes in ECs: the MAPK-ERK gene was higher in cells responding to the DAE group, and even higher in the H_Adip + DAE group. However, there was no difference in the H_Adip + nHA group when compared to Ctrl (A). The JNK was down-regulated in the H_Adip group when compared to Ctrl, and higher in response to DAE treatment when compared to H_Adip. Data are reported as means ± standard deviations (n = 3). Comparison by one-way ANOVA. Statistical differences were considered when * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, represented by black * when compared to the Ctrl group, by red * when compared to the H_Adip group, and by green * when compared between the groups with DAE.

Figure 8

c-Src involvement. After exposing the ECs to different shear-stress treatments for 72 h, the samples were obtained to perform the Western blotting assay and evaluate SRC protein content (A,B). The high adipogenesis condition increased the content of SRC in ECs, significantly in the H_Adip + DAE group, and less significantly in the H_Adip + nHA group. β-Actin was considered the protein loading control. Differences were considered significant when * p < 0.05, and *** p < 0.001, represented by black * when compared to the Ctrl group, and by red * when compared to the H_Adip group.

Figure 9

IL6 and IL1B gene expression changed in response to conditioned mediums. The samples were harvested as described earlier and the gene expression was measured using qPCR. To address the inflammatory effect of different conditions on ECs, we evaluated IL-6 (A) and IL-1β (B) genes. The 18SrRNA gene was considered the housekeeping gene and used to normalize the expression values. Data are reported in means ± standard deviations (n = 3). Differences were considered significant when * p < 0.05, *** p < 0.001, and **** p < 0.0001, represented by black * when compared to the Ctrl group, by red * when compared to the H_Adip group, and by green * when compared between the groups with DAE or between the groups with nHA. DAE = titanium with Dual Acid-Etching; nHA = titanium with nano-Hydroxyapatite-coated surface.