3D printed titanium anodized effects on human gingival fibroblasts response and bacterial colonization: a dual approach

This study demonstrates that anodizing 3D-printed (SLM) Ti6Al4V titanium surfaces enhances human gingival fibroblast adhesion and soft tissue integration without increasing bacterial colonization, offering a promising strategy for improving implant-supported prosthesis survival.

The Gist

The Big Picture: The "Race for the Surface"

Imagine you have just gotten a new titanium tooth implant. This implant sticks out of your gum, acting as a bridge between your jawbone and your fake tooth.

There is a constant, invisible race happening on the surface of this implant:

1. The Good Guys (Your Cells): Your body wants to send "construction workers" (gingival fibroblasts) to build a tight, protective seal around the implant so nothing bad can get in.
2. The Bad Guys (Bacteria): Harmful bacteria want to crash the party, build a sticky fortress (biofilm), and cause infection (peri-implantitis), which can make the implant fail.

The Problem: Currently, the surface of these implants is like a smooth, polished marble floor. It's too slippery for the construction workers to get a good grip, but it's just smooth enough that the bacteria can still slide right in and set up camp.

The Goal: The researchers wanted to create a surface that acts like Velcro for your good cells (so they stick tight) but like Teflon for the bad bacteria (so they can't stick at all).

---

The Experiment: 3D Printing Meets Nano-Tech

The team used a high-tech 3D printing method called Selective Laser Melting (SLM) to create the implant parts. This is like using a super-precise laser to melt metal powder and build a custom shape, rather than carving it out of a block.

Once they had the 3D printed metal, they didn't just polish it. They gave it a special chemical bath called Anodization. Think of this as giving the metal a "nano-skin."

The Result: This process created millions of tiny, hollow tubes on the surface, called Nanotubes.

• Size: These tubes are about 100 nanometers wide. To visualize this, if a nanotube were the size of a soda straw, a human hair would be as wide as a highway.
• Shape: They look like a microscopic honeycomb or a field of tiny straws standing up.

---

The Test: Who Wins the Race?

The researchers put human gum cells and a common mouth bacteria (Streptococcus gordonii) on two types of surfaces:

1. The Control: A standard, polished metal surface (what you get in a dentist's office today).
2. The New Stuff: The 3D printed metal with the nano-tubes.

1. How did the Human Cells react? (The Construction Workers)

• On the Standard Surface: The cells were okay, but they didn't hold on very tightly. If you tried to wash them off with a gentle enzyme (like a mild soap), many of them fell off.
• On the Nano-Tube Surface: The cells loved it! They spread out, grew long "fingers" (called filopodia) that grabbed onto the edges of the tiny tubes, and locked themselves in place.
• The Analogy: Imagine trying to walk on a smooth ice rink (Standard Surface) vs. walking on a grassy field with tall blades of grass (Nano-Tubes). On the grass, your feet can grab the blades, giving you a super-strong grip. The cells actually started producing more "glue" (a protein called Vinculin) to hold on even tighter.

Verdict: The new surface helped the body's cells build a much stronger, healthier seal.

2. How did the Bacteria react? (The Invaders)

• The Fear: The researchers were worried that making the surface "rough" with tubes might give the bacteria more places to hide, like a jungle gym for germs.
• The Result: Surprisingly, the bacteria didn't care. They stuck to the nano-tube surface just as much (or just as little) as they did to the smooth surface.
• The Analogy: The bacteria are like tiny, flat stickers. Whether the wall is smooth or has tiny straws sticking out, the stickers just don't have the right shape to grab onto the straws effectively. The "roughness" wasn't rough enough to trap them, but it was just right for the human cells.

Verdict: The new surface did not make the bacteria grow faster. It kept the "bad guys" in check while helping the "good guys."

---

Why This Matters

This study is a big deal because it solves a major headache in dentistry:

• Old Way: We had to choose between a surface that was safe for bacteria (smooth) but bad for cell attachment, or a surface that was good for cells but might trap bacteria.
• New Way: This 3D printed, nano-tube surface seems to have the best of both worlds. It encourages your body to heal and seal the implant tightly, without inviting a bacterial party.

The "Gold" Bonus

One fun side note: When they did this chemical process, the metal turned a yellow/gold color. This is actually a bonus! It means the implant could look more natural under the gum line, hiding the silver color of the titanium, which is great for the aesthetics of your smile.

The Bottom Line

The researchers found a way to 3D print a tooth implant part that has a microscopic "velcro" texture. This texture helps your gum cells grab on tight and heal properly, while the bacteria just slide right off. It's a promising step toward implants that last longer and stay healthier for life.

Technical Summary

1. Problem Statement

The long-term success of implant-supported prostheses (ISPs) relies not only on osseointegration (bone bonding) but also on soft tissue integration (STI) or "mucointegration." A major clinical complication is peri-implantitis, often triggered by bacterial plaque accumulation on the transmucosal abutment.

• Current Limitation: Standard clinical practice involves polishing titanium abutments to a mirror finish (Ra < 0.2 µm) to minimize bacterial adhesion. However, this smooth surface fails to promote strong adhesion of human gingival fibroblasts (HGFs), resulting in a weak connective tissue seal that is prone to bacterial invasion.
• The Challenge: There is a need for a surface modification strategy that simultaneously enhances HGF adhesion (to create a tight biological seal) while preventing bacterial colonization.
• Technological Gap: While Selective Laser Melting (SLM) allows for the creation of customized, complex implant geometries, few studies have evaluated the biological response of SLM-fabricated titanium surfaces that have been pre-polished (mimicking clinical laboratory protocols) and then anodized to create nanotubes.

2. Methodology

The study employed a dual-approach in vitro design, evaluating both eukaryotic (human cells) and prokaryotic (bacteria) responses.

• Sample Preparation:
  • SLM Group: Ti6Al4V discs were manufactured using Selective Laser Melting (SLM), followed by a heat treatment and a specific polishing protocol (using dental burs and polishing paste) to mimic clinical finishing. These were then subjected to electrochemical anodization (40V, 50 min, NH4F electrolyte) to create titanium nanotubes (NTs).
  • Control Group (MP-CTRL): Conventionally milled and polished Ti6Al4V discs (Grade 5) treated with the same finishing protocol but without anodization.
• Surface Characterization:
  • Morphology: Scanning Electron Microscopy (SEM) to measure nanotube diameter, wall thickness, and length.
  • Topography: 3D optical analysis for arithmetic average roughness (Ra) and surface roughness (Sa).
  • Wettability: Water contact angle measurements.
  • Protein Adsorption: Quantification of adsorbed salivary proteins and Bovine Serum Albumin (BSA).
• Biological Assays (Human Gingival Fibroblasts - HGFs):
  • Viability & Proliferation: Assessed at days 1, 4, and 7 using Live/Dead staining and cell counting.
  • Adhesion Strength: Resistance to enzymatic detachment (trypsinization) measured at 6h and 36h.
  • Morphology & Cytoskeleton: SEM imaging of filopodia; Immunofluorescence staining for vinculin (a focal adhesion protein) and F-actin to observe cell alignment and distribution.
  • Gene Expression: RT-qPCR analysis of Vinculin (VCL) gene expression at 48 hours.
• Bacteriological Assay:
  • Model Organism: Streptococcus gordonii (an early oral colonizer).
  • Protocol: Biofilm formation over 24 hours followed by sonication and Colony Forming Unit (CFU) counting to quantify bacterial adhesion.

3. Key Contributions

• Clinical Relevance: This is one of the first studies to evaluate anodized surfaces on SLM-fabricated titanium that has undergone a clinical-grade polishing protocol prior to nanostructuring. Most previous studies used machined or unpolished SLM surfaces.
• Dual-Approach Validation: The study uniquely assesses the "race for the surface" by simultaneously testing cell integration and bacterial colonization on the exact same surface modifications.
• Aesthetic Discovery: The authors noted that the anodization process imparted a yellow/gold color to the discs, which could offer an aesthetic advantage by masking the underlying grey titanium beneath the gingiva.

4. Key Results

• Surface Characteristics:

  • Anodization successfully created nanotubes with a diameter of ~100 nm, wall thickness of 15 nm, and length of ~500 nm.
  • Roughness: The nanotubular structure did not significantly alter the micrometric roughness; Ra remained ~0.2 µm (within the clinical threshold for low bacterial adhesion).
  • Wettability: Anodized surfaces (SLM-ANO) became significantly more hydrophilic (Contact Angle 29.5°) compared to polished controls (63–73°).
  • Protein Adsorption: No significant difference was found in the adsorption of salivary proteins or BSA between the groups.

• Cellular Response (HGFs):

  • Cytocompatibility: Both surfaces supported high cell viability (>95%) and similar proliferation rates.
  • Adhesion Strength: HGFs on SLM-ANO surfaces showed significantly higher resistance to enzymatic detachment (trypsin) at both 6h and 36h compared to controls.
  • Morphology: Cells on SLM-ANO exhibited numerous filopodia anchoring directly to the nanotube edges.
  • Vinculin Distribution: On SLM-ANO, vinculin was uniformly distributed across the cell membrane, whereas on controls, it was restricted to cell edges.
  • Gene Expression: Vinculin (VCL) gene expression was upregulated on SLM-ANO surfaces.
  • Alignment: Cells on both SLM-ANO and MP-CTRL showed uniform alignment (guided by polishing grooves), unlike random alignment on plastic.

• Bacterial Response:

  • S. gordonii Adhesion: There was no significant difference in bacterial colonization (CFU/mm²) between the anodized SLM surfaces and the polished controls. The nanotubes did not inhibit the early colonizer.

5. Significance and Conclusion

The study concludes that SLM-manufactured, polished, and anodized Ti6Al4V surfaces offer a promising solution for dental abutments:

1. Enhanced Soft Tissue Integration: The nanotubular structure significantly improves the adhesion strength and focal adhesion formation of human gingival fibroblasts without compromising cell viability.
2. Controlled Bacterial Risk: Crucially, this enhancement in cell adhesion did not come at the cost of increased bacterial adhesion. The surface maintained the low bacterial colonization levels associated with the standard polished control.
3. Clinical Viability: The combination of SLM (for customization), pre-polishing (for clinical relevance), and anodization (for bioactivity) creates a reproducible surface that promotes the "race for the surface" in favor of host cells.

Future Directions: The authors note that further studies are needed to include human epithelial cells (to assess the full soft tissue barrier) and multi-species bacterial biofilms to better simulate the complex oral ecosystem.