Journal of Biomedical Engineering and Biosciences (JBEB)

ISSN: 2564-4998

1. Introduction

Metallic biomaterials have attracted growing interest in biomedical engineering due to their excellent mechanical strength and acceptable biocompatibility. Their clinical use is especially common in orthopedic and dental implants, where durability and load-bearing capacity are essential [1]-[3]. Widely adopted examples include 316L stainless steel (SS), cobalt–chromium alloys, titanium alloys, and nickel–titanium alloys, all of which are valued for their favorable mechanical performance [4]-[6]. Despite this broad use, significant drawbacks remain, such as limited corrosion resistance, ion release, poor osseointegration, and mismatch in elastic modulus with bone tissue [7], [8]. These limitations highlight the urgent need for advanced metallic biomaterials with improved biological and mechanical properties.

High-entropy alloys (HEAs) have recently been introduced as promising candidates to overcome these challenges. Among them, the TiTaHfNbZr system combines mechanical robustness with strong potential for biocompatibility, making it suitable for biomedical applications [9]. Since implant surface characteristics directly influence cellular behavior and osseointegration, surface modification has become a key research focus. Femtosecond laser treatment offers precise control over surface topography while preserving bulk material properties, providing an effective route to enhance biological interactions [10]. Femtosecond laser surface texturing (FLST) has emerged as an effective technique for modifying implant surfaces at micro- and nano-scales, enhancing cell adhesion, alignment and promoting osseointegration. Recent studies have demonstrated that femtosecond laser-treated titanium and titanium alloy surfaces promote both cell adhesion and osteoblast migration [11, 12, 13]. For instance, Lackington et al. reported that bio-inspired micro- and nano-scale features produced on TiZr implants via femtosecond laser texturing significantly enhanced osseointegration [11]. Similarly, hierarchical laser-induced surface structures have been shown to guide osteoblast migration and support biological activity [12]. These findings highlight the clinical potential of FLST in implant surface modification and underscore its growing relevance in recent literature [13].

The present study investigates the biocompatibility and osseointegration potential of TiTaHfNbZr HEA and examines the role of femtosecond laser surface modification. This work provides a novel evaluation of how femtosecond laser–induced micro/nano-structured HEA surfaces influence cellular responses, including cell proliferation and apoptosis. Unlike previous studies, which have not studied the biological performance of laser-treated TiTaHfNbZr HEA in such detail, this research aims to clarify its biofunctional potential. Comparative evaluation with 316L stainless steel, a well-established biomedical alloy, is carried out to underline the advantages of HEA and demonstrate its potential for future orthopedic implant applications.

2. Methodologies

2.1. Sample Preparation

TiTaHfNbZr high-entropy alloy (HEA) with equiatomic composition was fabricated and cut into small samples, followed by grinding and polishing to standardize surface properties. This study provides one of the first biocompatibility evaluations of this material with a novel approach. 316L stainless steel (SS), a well-characterized orthopedic material, was used as a reference. Both HEA and SS specimens were prepared and characterized for surface morphology prior to biological testing.

In this study, among the designed laser surface patterns, the indent form pattern with a 50 µm spacing was selected for surface modification and biological evaluation. This specific topography represents a distinct configuration within the overall experimental design, allowing for a more focused assessment of its effect on cell behavior. Consequently, three groups were prepared: untreated SS, untreated HEA, and laser-patterned HEA (HEA-modified) for comparative analysis.

2.2. Surface Characterization

Surface characterization of control and laser-treated samples was initially performed using profilometry to determine average roughness (Ra) values and ensuring similar surface properties of HEA-control and SS samples.

2.3. Cell-material interactions

Cellular responses were evaluated to investigate the effects of material type and surface characteristics. Human bone osteosarcoma cells (Saos-2, ATCC HTB-85) were used throughout the cell-material interaction tests. Cells were cultivated in DMEM medium supplemented with 15% fetal bovine serum, 1% penicillin/streptomycin, and 1% GlutaMAX (Sigma Aldrich, product no: D6546). Cell proliferation was assessed using the 3-(4,5 Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay following a 3-day incubation period, in order to evaluate how both material composition and surface modification influenced proliferative activity. Apoptosis was analyzed using the Annexin V/Propidium Iodide (PI) staining protocol under the same conditions to determine the impact of material type and femtosecond laser treatment on cell viability. Finally, cell–material interactions were examined through cell fixation and imaging with scanning electron microscopy (SEM), enabling detailed investigation of cell morphology, adhesion, and spreading on the different surfaces. This combined approach provided a comprehensive assessment of proliferation, apoptosis, and cell–material interactions, allowing comparison between stainless steel, untreated HEA, and HEA-modified samples.

3. Results

Sample surfaces were first characterized using profilometry. According to the profilometer analysis, average surface roughness values of the control samples (SS and HEA-control) were found to be quite similar (±5%). In contrast, HEA-modified sample exhibited a significant increase in roughness—approximately 20-fold—compared to control samples (Figure 1).

The MTT assay, performed to evaluate the influence of material type and surface features on cellular behaviour, demonstrated that the HEA-control samples induced approximately 2.5-fold higher cell proliferation per mm² compared to stainless steel. Moreover, laser-patterned HEA (HEA-modified) samples exhibited an additional ~40% increase in proliferation relative to HEA-control, with differences confirmed as statistically significant at the 95% confidence level using Tukey’s significance test. (Figure 2).

In the apoptosis assay, not only HEA-control but also HEA-modified samples exhibited approximately 20% higher proportions of viable cells compared to stainless steel. Additionally, femtosecond laser patterning did not negatively affect cell apoptosis rates as compared to the control group of HEA (Figure 3).

SEM analysis of cell–material interactions revealed that laser-patterned HEA samples displayed enhanced cell attachment and broader surface coverage compared to untreated HEA-control. Furthermore, among the control groups, HEA surfaces showed higher cell density and more extensive cellular extensions on the material surface (Figure 4).

These findings are consistent with previous reports on femtosecond laser surface texturing, which have shown to enhance cell adhesion, guide osteoblast migration, and promote osseointegration on titanium-based implants [11–13]. These results suggest that the femtosecond laser–modified TiTaHfNbZr HEA surface supports improved early cellular responses compared to control groups. Such improvemnets are known to facilitate faster osseointegration and more stable implant–bone interfaces, which could potentially contribute to reduced healing time and lower revision risk in orthopedic applications.

5. Conclusion

The findings demonstrate that the TiTaHfNbZr high-entropy alloy offers substantially improved biocompatibility compared to 316L stainless steel, as evidenced by enhanced cellular proliferation, stronger adhesion, and higher viability. Surface modification via femtosecond laser patterning further amplified these effects by promoting favorable cell–material interactions and more extensive surface coverage. Collectively, the results suggest that elemental composition of high entropy alloy and laser surface patterning not only enhances intrinsic biocompatibility but also supports superior osseointegration, underscoring its significant potential as an innovative material for next-generation orthopedic implants.

References