Novel Ti6Al4V Surface Treatment for Subperiosteal Dental Implants (Part II): Extracellular Matrix Deposition and Osteogenic Marker Expression

Valentina Schiavoni, Lucia Memé, Giovanni Tossetta, Daniela Marzioni, Fabrizio Bambini, Andrea Frontini, Chiara Santoni, Paolo Moretti, Arianna Vignini, Roberto Campagna, Eleonora Salvolini.

Full text link: https://pubmed.ncbi.nlm.nih.gov/42073689/

Novel Ti6Al4V Surface Treatment for Subperiosteal Dental Implants (Part II): Extracellular Matrix Deposition and Osteogenic Marker Expression

1. Scientific Reference

• Study Title: Novel Ti6Al4V Surface Treatment for Subperiosteal Dental Implants (Part II): Matrix Deposition and Osteogenic Markers
• Authors: Valentina Schiavoni, Lucia Memé, Giovanni Tossetta, Daniela Marzioni, Fabrizio Bambini, Andrea Frontini, Chiara Santoni, Paolo Moretti, Arianna Vignini, Roberto Campagna, Eleonora Salvolini
• Journal: Materials
• Publication Year: 2026
• DOI: 10.3390/ma19081522

2. Scientific Background

The resurgence of patient-specific subperiosteal implants has created renewed interest in biomaterials capable of improving the biological interface between titanium devices and bone. Advances in cone-beam computed tomography (CBCT), digital treatment planning, CAD/CAM design and additive manufacturing have enabled the production of custom-made titanium frameworks for patients with severe alveolar bone atrophy, particularly when conventional endosseous implants may require extensive regenerative procedures.

While modern manufacturing technologies have significantly improved implant design accuracy, the biological behavior of implant surfaces remains a critical determinant of long-term success. Surface modifications are increasingly investigated to enhance osteoblast activity, accelerate osseointegration and promote stable bone formation. However, many experimental studies focus primarily on early cellular events such as adhesion and proliferation, whereas extracellular matrix maturation and mineralization—both essential for establishing a durable bone-implant interface—have received comparatively less attention.

This study represents the second phase of a research project evaluating a proprietary surface treatment applied to laser-melted Ti6Al4V substrates. Building upon previous findings demonstrating favorable early osteoblastic responses, the present investigation examines whether this novel surface also supports later stages of osteogenic differentiation associated with extracellular matrix maturation.

3. Study Objective

The primary objective of this investigation was to evaluate whether a proprietary surface treatment developed for Ti6Al4V subperiosteal dental implants promotes advanced osteogenic maturation beyond the initial stages of cell adhesion and proliferation.

Specifically, the authors assessed extracellular matrix formation, collagen organization, mineral deposition and the expression of key osteogenic proteins, including collagen type I (COL1A1), SPARC and dentin matrix protein 1 (DMP1). Through this approach, the study aimed to provide a more comprehensive biological characterization of the treated titanium surface and to clarify its potential relevance for next-generation custom-made subperiosteal implant systems.

4. Methodology

This research is a preclinical in vitro study performed using the human osteoblast-like MG-63 cell line.

Cells were cultured directly on Grade 5 Ti6Al4V titanium discs prepared with five different surface conditions: untreated control (CTRL), electroerosion polishing (EE), combined etching and sandblasting (ES), color anodizing (CA), and a proprietary surface treatment specifically developed for subperiosteal implant applications (ATcs).

Multiple complementary analytical techniques were employed to investigate the biological response:

• Atomic Force Microscopy (AFM) to characterize surface roughness;
• Scanning Electron Microscopy (SEM) combined with Energy Dispersive X-ray Spectroscopy (EDS) to evaluate cell morphology, adhesion and mineralized deposits;
• Western blot analysis to quantify the expression of DMP1, COL1A1 and SPARC;
• Immunofluorescence and confocal microscopy to determine the intracellular localization of DMP1.

Biological experiments were performed using a blinded design to minimize observational bias. Because this was an in vitro investigation, no patients, clinical implants or follow-up periods were involved.

5. Main Findings

All tested Ti6Al4V surfaces demonstrated good biocompatibility by supporting MG-63 cell attachment and preserving normal cellular morphology. However, clear differences emerged in the biological responses induced by the various surface treatments.

Among all experimental groups, the proprietary ATcs surface exhibited the most distinctive osteogenic profile. Scanning electron microscopy revealed a continuous layer of flattened, polygonal osteoblast-like cells that remained closely attached to the titanium substrate, suggesting strong cell-surface interactions. In addition, SEM identified extracellular mineral-like nodules surrounding the cells. Elemental analysis using EDS confirmed that these deposits were enriched in calcium and phosphorus, with a calcium-to-phosphorus ratio consistent with hydroxyapatite, indicating active extracellular matrix mineralization rather than nonspecific precipitation.

Protein expression analyses further supported these observations. DMP1, a recognized marker of late osteogenic differentiation, showed increased expression on ES, ATcs and CA surfaces compared with the untreated control. In contrast, COL1A1 expression was more pronounced on CTRL and EE surfaces and progressively decreased on ES and ATcs specimens. Likewise, SPARC expression was higher on EE and CA surfaces but comparatively lower on ATcs. According to the authors, this coordinated expression pattern is compatible with progression from early extracellular matrix synthesis toward a more mature mineralization phase rather than sustained proliferative activity.

Confocal immunofluorescence analyses confirmed abundant cytoplasmic DMP1 staining in cells cultured on ATcs discs, whereas only weak staining was observed on CTRL and EE surfaces. Taken together, these complementary findings suggest that the proprietary surface treatment promotes extracellular matrix maturation and mineral deposition in addition to supporting osteoblast viability. Nevertheless, the authors emphasize that these conclusions are limited to an in vitro experimental model and require validation through in vivo investigations before clinical implications can be established.

6. Clinical Analysis

This study extends the biological characterization of a novel Ti6Al4V surface treatment by examining events that occur after the initial stages of osteoblast attachment. Rather than focusing solely on cell proliferation, the authors investigated molecular and structural features associated with extracellular matrix maturation, an essential prerequisite for successful osseointegration.

The findings suggest that the ATcs surface creates a biological environment that favors osteogenic progression instead of simply stimulating early cellular activity. Increased DMP1 expression, together with the formation of calcium-phosphate-rich mineralized deposits and organized extracellular matrix, indicates that osteoblast-like cells cultured on this surface advance toward a more mature phenotype capable of supporting mineralization. Importantly, the reduced expression of COL1A1 and SPARC should not be interpreted as diminished biological performance. Within the context of osteoblast differentiation, these proteins are typically associated with earlier stages of matrix production and remodeling, making their coordinated reduction alongside increased DMP1 biologically consistent with later osteogenic maturation.

From a clinical perspective, these observations are particularly relevant for custom-made subperiosteal implants. Unlike conventional endosseous implants, these devices rely predominantly on intimate contact with the cortical bone surface rather than deep intrabony anchorage. Consequently, surface characteristics capable of promoting extracellular matrix maturation may contribute to establishing a more favorable biological interface between bone and implant.

Despite these promising biological findings, caution is warranted. The study does not evaluate implant survival, biomechanical stability, bone remodeling under functional loading, or clinical outcomes. Furthermore, the proprietary composition of the ATcs treatment remains confidential, limiting independent assessment of the physicochemical mechanisms responsible for the observed effects. As acknowledged by the authors, further animal studies, biomechanical investigations and clinical trials are required before these laboratory observations can be translated into evidence-based clinical recommendations.

7. Clinical Applications

The findings of this study are primarily relevant to the development of patient-specific subperiosteal dental implants manufactured from Ti6Al4V using digital workflows that combine CBCT imaging, computer-aided design and additive manufacturing.

If future in vivo studies confirm the biological responses observed in this investigation, the proprietary ATcs surface treatment could represent a promising strategy for improving the biological interface between titanium implants and cortical bone. This may be particularly valuable in patients presenting with severe alveolar bone atrophy, where subperiosteal implants offer an alternative to extensive bone augmentation procedures.

The study also supports continued research into implant surface engineering aimed at enhancing extracellular matrix maturation rather than solely accelerating early osteoblast proliferation. Such an approach could contribute to the design of next-generation implant surfaces intended to optimize biological integration.

However, it is important to emphasize that this study does not demonstrate improved implant survival, reduced complication rates, faster healing, or superior clinical performance. The results should therefore be viewed as biological evidence supporting further preclinical and clinical research rather than as proof of clinical efficacy.

8. Level of Evidence, Limitations and Transparency

This publication represents a preclinical in vitro investigation, providing mechanistic insight into osteoblast behavior on differently treated Ti6Al4V surfaces. While this type of research is valuable for understanding biological interactions at the bone–implant interface, it cannot predict clinical performance or long-term implant success in human patients. Consequently, the overall level of evidence should be considered limited when compared with animal studies or clinical trials.

One strength of the study is the use of multiple complementary analytical techniques—including AFM, SEM-EDS, Western blotting and confocal microscopy—which consistently support the observed biological responses. Nevertheless, several limitations should be acknowledged. The investigation was conducted exclusively on an osteoblast-like cell line, without animal validation, biomechanical testing, or clinical evaluation. Furthermore, the observation period was limited to early laboratory experiments and did not assess long-term tissue responses.

The authors report no external funding and declare no conflicts of interest. They also indicate that the detailed composition of the proprietary ATcs surface treatment remains confidential due to industrial restrictions. While this limits complete reproducibility of the manufacturing process, it does not alter the experimental observations reported in the study. The authors explicitly state that additional in vivo investigations will be necessary before drawing conclusions regarding clinical osseointegration.

9. Key Points

• Study type: Preclinical in vitro experimental study.
• Level of evidence: Low to moderate (fundamental laboratory research).
• Study model: Human osteoblast-like MG-63 cell line.
• Number of patients: Not applicable.
• Number of implants: Not applicable.
• Observation period: 72-hour cell culture for the primary biological analyses.
• Material investigated: Grade 5 Ti6Al4V titanium discs with five different surface treatments (CTRL, EE, ES, ATcs and CA).
• Primary outcome: Evaluation of extracellular matrix formation and osteogenic differentiation through DMP1, COL1A1 and SPARC expression.
• Main finding: The proprietary ATcs surface promoted a biological profile characterized by increased DMP1 expression, organized extracellular matrix deposition and calcium-phosphate-rich mineralized nodules, consistent with advanced osteogenic maturation.
• Scientific conclusion: The findings support the biological potential of the ATcs surface treatment for enhancing late-stage osteogenic events associated with bone–implant interface formation in custom-made subperiosteal implants.
• Study limitations:
  • In vitro experimental design.
  • No animal or human clinical validation.
  • No biomechanical or long-term performance assessment.
  • Proprietary surface treatment not fully disclosed.

10. Scientific Impact

This study contributes to the growing body of research investigating how implant surface engineering can influence the biological events that ultimately determine successful osseointegration. Rather than concentrating exclusively on early osteoblast adhesion or proliferation, the authors examined extracellular matrix maturation and mineralization, providing a more comprehensive understanding of how surface modifications may regulate osteogenic progression.

An important aspect of this work is its continuity with the authors’ previous publication, which demonstrated favorable early cellular responses to the same proprietary Ti6Al4V surface treatment. By extending the investigation to later stages of osteoblast differentiation, the present study strengthens the biological rationale supporting this surface technology for patient-specific subperiosteal implants.

Although the results remain limited to laboratory conditions, they identify DMP1 expression, extracellular matrix organization and calcium-phosphate deposition as complementary biological indicators of advanced osteogenic activity. These observations provide a foundation for future translational research evaluating whether these molecular and structural responses translate into improved bone–implant integration under physiological conditions.

Rather than establishing a new clinical standard, this investigation serves as an important preclinical step toward validating innovative surface treatments for digitally manufactured subperiosteal implant systems.

11. Editorial Conclusion

This preclinical study provides a detailed biological evaluation of an innovative Ti6Al4V surface treatment developed for custom-made subperiosteal dental implants. The coordinated increase in DMP1 expression, organized extracellular matrix deposition and formation of calcium-phosphate-rich mineralized structures suggests that the ATcs surface supports advanced osteogenic maturation beyond the early phases of cell attachment and proliferation. While these findings strengthen the biological rationale for this surface technology, they remain restricted to an in vitro model. Well-designed animal studies and clinical investigations are required to determine whether these promising laboratory observations ultimately translate into improved osseointegration and long-term clinical outcomes.