Advancing osseointegration research: A dynamic three-dimensional (3D) in vitro culture model for dental implants

Abstract

Background/purpose: In-vitro studies are essential for understanding cellular responses, but traditional culture systems often neglect the three-dimensional (3D) structure of real implants, leading to limitations in cellular recruitment and behavior largely governed by gravity. The objective of this study was to pioneer a novel 3D dynamic osteoblastic culture system for assessing the biological capabilities of dental implants in a more clinically and physiologically relevant manner.

Materials and methods: Rat bone marrow-derived osteoblasts were cultured in a 24-well dish with a vertically positioned dental implant. Controlled rotation using a 3D rotator with 3° tilts was applied. Cell attachment, proliferation, and differentiation on implant surfaces were evaluated in response to different surface topographies, physicochemical properties, and local environments.

Results: Among the tested rotational speeds (0, 10, 30, 50 rpm), optimal osteoblast attachment and proliferation were observed at 30 rpm. A linear correlation was found between cell attachment and rotation speed up to 30 rpm, declining at 50 rpm. Alkaline phosphatase (ALP) activity and mineralized matrix formation were elevated on newly acid-etched, hydrophilic surfaces compared to their 4-week-old hydrophobic surfaces. Sandblasted implants showed higher ALP activity and matrix mineralization. Adding N-acetyl cysteine to the culture medium increased ALP activity and mineralization.

Conclusion: Osteoblasts successfully attached, proliferated, and mineralized on dental implants in vitro under optimized dynamic conditions. This system differentiated the biological capabilities of implants with varying surface topographies, wettability, and biochemically modulated environments. These findings support developing a 3D dynamic dental implant culture model, advancing osseointegration research and innovating dental implant designs.

Keywords: Hydrophilic materials; Osseointegration; Osteoblasts; Surface properties.

Figure 1

Study design. (A) Schematic illustration of the experimental setup designed to determine the optimal rotation speed for enhancing osteoblast attachment and proliferation on dental implant surfaces vertically positioned at the center of the well. (B) Overview of the experimental strategy to assess the sensitivity of the three-dimensional (3D) dynamic culture system. The sensitivity of the 3D culture system to discern three key factors—1) surface morphology, 2) wettability, and 3) biochemical supplements—was evaluated by examining osteoblast differentiation.

Figure 2

A dynamic three-dimensional culture model for dental implants. (A) A dental implant standing upright was secured using 0.002 g of resin bonding cement in a well of a 24-well cell culture-grade polystyrene dish. (B) Dental implants were fully immersed in the culture medium. (C) The culture dish underwent continuous rotational motion with 3° tilts and variable speed.

Figure 3

Surface characterization of dental implants. (A) Scanning electron microscopy (SEM) images of sandblasted and acid-etched implants are shown, including both low- and high-magnification views. (B) Quantitative assessment of surface roughness. An average roughness (Ra) and peak-to-valley roughness (Rz) parameters of sandblasted and acid-etched implant surfaces, along with representative images of roughness profiles. (C) Creation of hydrophilic and hydrophobic implants with identical surface morphology. Wettability evaluation for new and old acid-etched implants is shown. The “new” surface represents the implant immediately after acid-etching, while the “aged” surface represents a 4-week-old implant after acid-etching (stored for 4 weeks in a dark condition). Both surfaces had identical surface topography as shown in Fig. 3A. Representative images show a 3 μL water droplet placed on the surfaces with a histogram of measured contact angles. Data are presented as mean ± standard deviation (n = 3). ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Figure 4

Osteoblast attachment efficiency in three-dimensional dynamic culture under different rotational speeds. (A) Quantification of osteoblasts attached to implant surfaces under various rotational conditions, assessed by the tetrazolium salt-based colorimetric assay (WST-1) on day 2 of culture. Data are presented as mean ± standard deviation (n = 3). ∗P < 0.05. (B) Fluorescence microscopy images showing the distribution of osteoblasts across three different zones of the implant surfaces after day 2 under varied rotational speeds. Cells are dual-stained, with Rhodamine-phalloidin (red) for actin filaments and 4′,6-diamidino-2-phenylindole (DAPI) (blue) for nuclei. Scale bar: 320 μm.

Figure 5

Osteoblast proliferation in response different rotational speeds. (A) The number of propagated osteoblasts on implant surfaces on days 4 and 6 under different rotational speeds. Data are presented as mean ± standard deviation (n = 3). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗∗P < 0.0001. (B) Growth curve illustrating osteoblast proliferation over time, measured at days 2, 4, and 6 of culture.

Figure 6

Influence of rotation speed on osteoblast attachment in three-dimensional dynamic culture. The graph illustrates the relationship between rotation speed and the number of osteoblasts attached to implant surfaces, plotted separately for days 2 and 4 of culture. Regression analysis revealed a linear correlation when data at 50 rpm were excluded, and a quadric curve when all data points were considered.

Figure 7

Influence of surface and environmental factors on osteogenic response. (A) Comparison of day 5 alkaline phosphatase (ALP) activity between sandblasted versus acid-etched implants. (B) Assessment of matrix mineralization by alizarin red staining on day 10 and representative images of sandblasted and acid-etched implants. (C) Comparison of ALP activity between hydrophobic versus hydrophilic implants. (D) Assessment of matrix mineralization and representative images of implants for both hydrophobic and hydrophilic surface. (E) Changes in ALP activity with or without N-acetylcysteine (NAC) supplementation to the culture medium. (F) Assessment of matrix mineralization and representative images of stained implants with and without NAC supplementation. Data are presented as mean ± standard deviation (n = 3). ∗P < 0.05. ∗∗P < 0.01. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.