ABSTRACT
Background:
The surface roughness of dental implants plays a critical role in osseointegration by enhancing bone-implant contact. However, the optimal surface topography to maximize bone response is still under investigation.
Materials and Methods:
Titanium implants with three different surface roughness categories were tested: smooth (Ra = 0.5 μm), moderately rough (Ra = 1.5 μm), and rough (Ra = 3.0 μm). The samples were incubated with osteoblast-like cells (MG-63) for 14 and 28 days. Alkaline phosphatase (ALP) activity, cell viability (MTT assay), and mineralization (Alizarin Red staining) were assessed to quantify the bone response. Scanning Electron Microscopy (SEM) was performed to examine cell morphology on the implant surfaces.
Results:
At 14 days, ALP activity was significantly higher on moderately rough surfaces (30.5 ± 1.2 U/L) compared to smooth (18.2 U/L ±0.8 U/L) and rough surfaces (22.6 U/L ±1.0 U/L). The MTT assay showed improved cell proliferation on moderately rough surfaces (optical density 0.85 ± 0.03) compared to smooth (0.56 ± 0.02) and rough surfaces (0.61 ± 0.04). Mineralization at 28 days was most prominent on moderately rough surfaces, with Alizarin Red absorbance of 0.95 ± 0.02, followed by rough (0.75 ± 0.03) and smooth (0.40 ± 0.02). SEM analysis revealed well-spread cell morphology on both moderately rough and rough surfaces.
Conclusion:
Moderately rough implant surfaces exhibited superior bone response compared to smooth and rough surfaces, highlighting the importance of controlled surface topography in enhancing osseointegration. Future studies should explore the molecular mechanisms underlying these differences to optimize implant design further.
KEYWORDS: Dental implants, osseointegration, surface roughness
INTRODUCTION
The success of dental implants largely depends on achieving stable osseointegration, defined as a direct and functional connection between the implant surface and the surrounding bone tissue. Implant surface modifications, particularly roughness, play a critical role in influencing biological responses at the bone-implant interface.[1] Research suggests that increased surface roughness enhances osteoblast adhesion, proliferation, and differentiation, promoting early-stage osseointegration.[2] Moderately rough surfaces, with an average roughness (Ra) between 1 μm to 2 μm, have shown better outcomes in terms of bone-implant contact compared to both smooth and excessively rough surfaces.[3,4]
Implant surface roughness impacts protein adsorption, influencing osteoblast behavior by modulating integrin signaling and cytoskeletal organization.[5] Additionally, rough surfaces increase the surface area, promoting mechanical interlocking between the implant and the bone.[6] However, very high roughness can increase the risk of bacterial colonization and peri-implantitis, which may compromise long-term stability.[7] Thus, optimizing the surface roughness is crucial for ensuring a balance between osseointegration and minimizing bacterial contamination risks.
In vitro studies provide essential insights into cellular responses to different surface topographies. They allow for controlled analysis of osteoblast proliferation, alkaline phosphatase (ALP) activity and mineralization, which are critical indicators of osseointegration potential.[8]
MATERIALS AND METHODS
Titanium Grade IV implants were prepared with three different surface roughness levels: smooth (Ra = 0.5 μm), moderately rough (Ra =1.5 μm), and rough (Ra = 3.0 μm). Surface roughness was measured using a profilometer (Mitutoyo SJ-210, Japan). The implants were sterilized through autoclaving at 121°C for 15 min before use. The osteoblast-like cell line MG-63 (ATCC, USA) was employed to assess bone response. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine, and incubated at 37°C in a humidified atmosphere with 5% CO2. Medium changes were performed every 48 hours. Implants were divided into three experimental groups based on surface roughness: Group A (smooth surface, Ra = 0.5 μm), Group B (moderately rough surface, Ra = 1.5 μm), and Group C (rough surface, Ra = 3.0 μm). MG-63 cells were seeded onto the implant surfaces at a density of 1 × 105 cells per implant, placed in 24-well plates with 1 mL of culture medium per well, and incubated for 14 and 28 days with media changes every two days.
Alkaline phosphatase (ALP) activity was measured on days 14 and 28 using a colorimetric p-nitrophenyl phosphate (pNPP) assay, with absorbance recorded at 405 nm (Bio-Rad, USA). Cell proliferation was assessed using the MTT assay, where MTT reagent (5 mg/mL) was incubated for 4 hours, and the resulting formazan crystals dissolved in DMSO with absorbance measured at 570 nm. Mineralization was evaluated at day 28 using Alizarin Red S staining, with absorbance quantified at 405 nm. For scanning electron microscopy (SEM), cells were fixed with 2.5% glutaraldehyde, dehydrated in a graded ethanol series, and sputter-coated with gold before imaging (Zeiss, Germany). All experiments were performed in triplicate (n = 3). Statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons, with significance set at P < 0.05 (SPSS version 27, IBM Corp., USA).
RESULTS
Alkaline Phosphatase (ALP) activity
ALP activity increased significantly over time in all groups, with the highest levels observed in the moderately rough surface group. At day 14, Group B (moderately rough) showed significantly higher ALP activity (30.5 U/L ±1.2 U/L) compared to Group A (smooth) (18.2 U/L ±0.8 U/L) and Group C (rough) (22.6 U/L ±1.0 U/L). By day 28, Group B maintained the highest ALP activity (50.8 ± 2.1 U/L), followed by Group C (35.2 U/L ±1.3 U/L) and Group A (25.1 U/L ± 1.0 U/L) [Table 1].
Table 1.
Alkaline phosphatase (ALP) activity
| Group | Day 14 (U/L) | Day 28 (U/L) |
|---|---|---|
| Smooth (Group A) | 18.2±0.8 | 25.1±1.0 |
| Moderately Rough (Group B) | 30.5±1.2 | 50.8±2.1 |
| Rough (Group C) | 22.6±1.0 | 35.2±1.3 |
Cell viability (MTT assay)
The MTT assay showed that cell viability improved over time on all surfaces. Group B demonstrated the highest cell viability at both 14 days (0.85 ± 0.03 OD) and 28 days (1.32 ± 0.05 OD). Group A had the lowest cell proliferation, with absorbance values of 0.56 ± 0.02 at day 14 and 0.85 ± 0.04 at day 28 [Table 2].
Table 2.
Cell viability (MTT assay)
| Group | Day 14 (OD) | Day 28 (OD) |
|---|---|---|
| Smooth (Group A) | 0.56±0.02 | 0.85±0.04 |
| Moderately Rough (Group B) | 0.85±0.03 | 1.32±0.05 |
| Rough (Group C) | 0.61±0.04 | 1.05±0.06 |
Mineralization (Alizarin red staining)
The highest mineralization was observed in Group B on day 28, with an absorbance value of 0.95 ± 0.02. Group C also showed substantial mineralization (0.75 ± 0.03), while Group A had the least mineralization (0.40 ± 0.02) [Table 3].
Table 3.
Mineralization (Alizarin red staining)
| Group | Alizarin Red Absorbance (Day 28) |
|---|---|
| Smooth (Group A) | 0.40±0.02 |
| Moderately Rough (Group B) | 0.95±0.02 |
| Rough (Group C) | 0.75±0.03 |
Scanning Electron Microscopy (SEM) observations
SEM analysis revealed distinct cell morphologies across the three groups. Cells on the smooth surface (Group A) appeared elongated with limited spreading. On the moderately rough surface (Group B), cells were well-spread with prominent filopodia and better surface coverage. The rough surface (Group C) also showed good cell attachment but with uneven spreading and clustered growth patterns.
DISCUSSION
The present study evaluated the bone response to titanium implants with varying surface roughness levels, highlighting that moderately rough surfaces (Ra = 1.5 μm) exhibited superior osteoblast activity, cell viability, and mineralization compared to both smooth and rough surfaces. These findings align with previous research, which demonstrates that surface topography significantly influences osseointegration by affecting cell attachment and proliferation.[1] Moderately rough surfaces provide optimal conditions for cellular responses, enhancing protein adsorption and integrin-mediated signaling necessary for osteoblast differentiation.[2]
The increased ALP activity observed on moderately rough surfaces corroborates previous reports, which emphasize the positive effect of such surfaces on early osteoblast differentiation.[3] ALP is a critical enzyme in bone mineralization, and its elevated levels indicate enhanced osteoblastic activity and early-stage osseointegration.[4] In comparison, smooth surfaces have been shown to inhibit osteoblast proliferation due to a reduced surface area and limited cell adhesion sites.[5] This finding is consistent with our data, where the smooth surfaces (Ra = 0.5 μm) exhibited the lowest ALP activity and mineralization.
Interestingly, excessively rough surfaces (Ra = 3.0 μm) did not outperform the moderately rough surfaces in terms of osteoblast function, despite the increased surface area. This result is consistent with findings from Wennerberg and Albrektsson,[6] who observed that while rough surfaces promote initial cell adhesion, extreme roughness can impair cell spreading and lead to uneven cell distribution. Moreover, excessively rough surfaces may also increase the risk of bacterial colonization, which could compromise implant longevity.[7,8,9] These findings emphasize the need to balance surface roughness to promote osseointegration without enhancing bacterial adherence.
CONCLUSION
In conclusion, this study reinforces that moderately rough surfaces (Ra = 1.5 μm) offer the most favorable conditions for osteoblast activity, cell proliferation, and mineralization. These findings suggest that implant manufacturers should consider optimizing surface roughness to enhance osseointegration while minimizing the risk of bacterial contamination.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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