A Clean, Rapid, and Controllable Approach for Establishing Bioactive Surfaces on Zirconia Implants

Zirconia implants have great application prospects due to their favorable biocompatibility and esthetic properties. However, the percentage of osseointegration of zirconia implants is lower than that of titanium implants. This study, structures bioactive surfaces on zirconia using a femtosecond laser combined with hydroxyapatite (HA) crystals and evaluates the surface morphology, functional groups, crystal phase, mechanical performance, and cell response. The results revealed that calcium phosphate deposition coincided with the establishment of micro‐grooves on the zirconia surface. The crystal composition and flexural strength of zirconia showed no significant changes after the surface treatment. Furthermore, the modified surface promoted cell adhesion and proliferation through adjustments in cell morphology. The findings suggest that the combination of femtosecond laser and HA crystals offers a clean, rapid, and controllable approach to preparing bioactive surfaces for zirconia implants.


Introduction
Yttria-stabilized tetragonal zirconia exhibits good chemical stability, high flexural strength and fracture toughness. [1]irconia implants, which are toothcolored and offer excellent esthetics have been reported to be less prone to bacterial colonization, [2,3] making them a suitable alternative to titanium (Ti) implants.However, zirconia implants have been criticized for their bio inertness and inadequate osteointegration, [4] the bone contact rate is lower compared to Ti implants. [5]Researchers have attempted various surface treatment techniques used for Ti implants on zirconia implants, such as sandblasting, [6] acid etching, [7] and laser treatment. [8]onsidering the drawbacks of methods like sandblasting (surface damage and lack of control), [9] and etching (surface contamination and potential toxicity), [10] laser texturing treatment is more suitable for surface modification of zirconia dental implants.Recently, the use of femtosecond laser for surface modification has gained popularity due to its extremely high instantaneous power, which can be precisely focused on micron-grade superfine space areas without causing significant damage. [11]14] In addition to microscopic roughness, the chemical composition of the implant surface is another important factor determining osseointegration.Calcium phosphate (CP) is the primary component of bone mineral.Materials based on CP, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), are known to be bioactive and capable of stimulating bone regeneration. [15]hese material coatings could promote the integration between bone tissue and the implant surface. [16][19] However, CP coatings exhibit poor stability and provide weak bonding strength to the zirconia substrate using the abovep-noted methods. [20]The coatings didn't provide roughed microstructure on the surface.
Therefore, one challenge is to simultaneously produce macro and nano microstructures while increasing the adhesion strength of the CP coating, without affecting the bulk properties of the zirconia implants.Femtosecond laser ablation is based on the nonlinear processes of light absorption and ionization unleashed by the effect of irradiation with very short and intense pulses, which produce an instant extremly high temperature and high impression condition. [11]Can any chemical reactions occur between the interfaces if some bioactive substances are lined on the zirconia surface during the femtosecond laser ablation?There has been no such study until now.
In order to achieve the simultaneous formation of desired microstructure and bioactive chemical constituents, the hypothesis of this study is to combine femtosecond laser and hydroxyapatite (HA) crystals to induce a rough microstructure and CP deposition on zirconia surface concurrently.The implementation of this study is helpful in establishing a clean, rapid and controllable surface treatment method for zirconia implants, which can promote the Osseo integration of zirconia implants and provide an experimental basis for further clinical application of zirconia implants.

Specimen Preparation
Yttria stabilized tetragonal zirconia (Upcera, Shenzhen, China) specimens (N = 36) with disk shape were prepared using computer-aided design and computer-aided manufacturing (CAD/CAM) techniques.The specimens were fully sintered according to the manufacturer's instructions.The surface of each specimen was then ground and polished using silicon carbide paper (600, 1200, and 1500 grit) for 20 min.The final specimens were consistent in dimension, with a diameter of 6 mm and thickness of 2 mm.

Surface Treatment
The prepared specimens were randomly divided into three groups.Group one (n = 12) served as the blank control group, where no surface treatment was applied after polishing (referred to as the CL group).Group two (n = 12) underwent femtosecond laser treatment (referred to as the FS group).The surface of each specimen in this group was treated solely with a femtosecond laser in the air.A titanium sapphire laser generator (Tsunami; Spectra-Physics, Santa Clara, CA, USA) and a corresponding regenerative amplification system (Spitfire; Spectra-Physics) were used to induce microstructure and enhance surface roughness.The laser beam had a pulse width of 120 fs, a central wavelength of 800 nm, and a repetition rate of 1 kHz.The pulse energy was 100 μJ and the scanning speed was 300 μm −1 s.
Group three (n = 12) constituted the experimental group, receiving femtosecond laser treatment combined with hydroxyapatite deposition (referred to as the FHA group).Prior to femtosecond laser treatment, a suspension of pure HA powder (Macklin, Shanghai, China) at a concentration of 8 mg mL −1 was prepared.HA powders were slowly added into deionized water and thoroughly stirred.A 30 μL droplet of hydroxyapatite suspension was applied to the surface of each sample and dried at 60 °C for 5 min to evaporate the solvent, forming a film of HA crystals on the surface of each specimen.Subsequently, the specimens underwent femtosecond laser treatment using the same output energy parameters as the FS group.The treatment process was illustrated in Figure 1.

The Surface Morphology Observation and Chemical Elements Examination
All the specimens were cleaned in deionized water for 10 min by an ultrasonic cleaner (SB-5200DT, SCIENTZ, China).The ultrasound frequency is 40 kHz and ultrasound the intensity is 300 W. Prior to imaging in a Scanning Electron Microscope (SEM, Quanta 650 FEG, FEI, Czech), a nanometric layer of palladium was sprayed onto the specimens.The surface morphology was observed using SEM and Laser confocal microscope (Zeiss Axio LSM700, Zeiss, Germany).The surface chemical elements were detected and analyzed using an energy-dispersive spectrometer (EDS, Quanta 650 FEG, FEI, Czech).

Surface Roughness Measurement
Surface roughness (Sa, Sq, Sz) of the specimens after different surface treatments was measured and surface morphology was photographed using a laser confocal microscope (Zeiss Axio LSM700, Zeiss, Germany, 1000×).Three separate areas were analyzed for each specimen and two specimens from each group were selected.

Surface Wettability Measurement
To assess the surface wettability, three specimens from each group were chosen for contact angle (CA) evaluation using droplet method with a high-temperature contact angle measuring instrument (Theta, Biolin, Finland).A controlled volume of 2 μL distilled water was dispensed each time by a computer, and images were captured once the droplet had stabilized.The contact angle was analyzed by curve fitting based on the photos.Two separate areas were analyzed for each specimen.

Analysis of Surface Functional Chemical Groups
The surface functional chemical groups of specimens from each group and hydroxyapatite powder were analyzed using a Fourier transform infrared spectrum analyzer (Nicolet iS50, Thermo, America).The wavelength range performed was 7800-350 cm −1 .

Crystal Phase Analysis
X-ray diffraction (XRD, D/MAX RAPID IIR, RIGAKU, Japan) measurements were conducted using a Copper target,  = 1.54056, voltage 47 kV, current 250 mA to investigate the crystal phase composition of the modified specimens and potential phase transformations resulting from laser processing.

Flexural Strength Test
Cuboid specimens with dimensions of 14 mm in length, 4 mm in width and 1.2 mm in thickness (N = 18) were fabricated using the same method as mentioned earlier and underwent surface treatment with different methods according to the aforementioned groups (n = 6 for the CL, FS, and FHA groups).All the specimens were placed in a universal testing machine (MTS Insight, Mechanical Testing America) and loaded until fracture occurred, following the ISO 6872-2015 standard. [21]A stainlesssteel ball with a diameter of 6 mm was applied to the specimens over a span of 12 mm at a speed of 0.5 mm min −1 .The flexural strength was calculated using the formula flexural strength = 3FL 2bhˆ2 −1 , [22] where F represents the maximum force (kN) at mid-span (1 kN = 1000 N), L denotes the span length (mm), b indicates the width (mm), and h represents the thickness (mm).

Cell Culture and Identification
Bone marrow mesenchymal stem cells (BMSCs) were isolated using the whole bone marrow adherence method from male Sprague Dawley (SD) rats aged 4 weeks, [23] following the guidelines and regulations of the Animal Care and Use Committee of the Central South University.BMSCs were purified and cultured in -minimum essential medium (-MEM; BI, Israel) supplemented with 10% fetal bovine serum (BI, Israel) and 1% penicillin/ streptomycin antibiotics (BI, Israel).Cell surface marker identification and multilineage differentiation potential identification were performed.
ing in 95% ethanol and deionized water for 10 min, respectively.After sterilizing at high temperature and high pressure, the specimens were transferred to a 48-well plate.BM-SCs were seeded at a density of 1 × 10 4 cells cm −2 onto the zirconia surface and cultured for 24 h.Subsequently, phosphate buffer saline (PBS; BI, Israel) was used to rinse the cells, followed by fixation using 4% paraformaldehyde (Biosharp, China) at room temperature.After three rinses, the specimens were dehydrated using a graded ethanol series, freeze-dried under vacuum for 1 h and sputter-coated with gold for SEM observation.

Statistics
Statistical analysis was conducted using SPSS v18.0 (SPSS, Chicago, IL), with a significance level set at p = 0.05.The results were analyzed statistically using a one-way analysis of variance (ANOVA).

Zirconia Surface Characterization Results
Well-distributed periodic micro-grooves were observed in the FS and FHA groups.These grooves exhibited neat edges, without any micro-cracks or fissures.Furthermore, granular material deposits were observed within the grooves of the FHA group (as indicated by the rectangle in Figure 2F,G).EDS analysis revealed a significant presence of calcium (Ca) and phosphorus (P) elements on the groove walls and the flat area between grooves of specimens from the FHA group (Figure 3C,D), and the Ca/P ratio is 1.5.Laser confocal microscope results demonstrated the 3D nature of the micro-groove (Figure 4).The micropattern appeared as a "V" shape with a narrow bottom and wide surface.Each groove had a width of ≈15 μm and a depth of ≈6 μm.The surface roughness for each subgroup is shown in Table 1.The surface roughness of both FS and FHA groups is statistically greater than the CL group (p < 0.05).
The CL group exhibited the lowest contact angle (CA) value (87.58°± 3.91°), followed by the FHA group (99.01°± 3.84°), and the FS group had the highest value (103.97°±8.02°) (Table 2).A statistical difference was observed between both the FS and FHA groups compared to the control group (p < 0.05).However, no statistical difference was found between the FS and the FHA groups.The FTIR results of all specimens are presented in Figure 5, revealing absorption peaks at 1250, 1420, 1456, 1602, and 3569 cm −1 , as well as two strong peaks at 1032 and 1090 cm −1 .The presence of the carbonate group is evident from peaks at 1456 and 1420 cm −1 .Furthermore, absorption peaks related to the bicarbonate group can be observed at 1250 and 1602 cm −1 .The peaks at 1090 and 1032 cm −1 in the FHA group demonstrate the existence of PO 4 3− ions, coinciding with the characteristic peaks observed in HA powder.The appearance of the peak at 3569 cm −1 corresponds to OH − .However, the FTIR spectrum of the FHA group does not show the absorption peaks related to the OH ─ bond.This indicates that femtosecond laser ablation can lead to the removal of OH − from hydroxyapatite crystals.

Crystal Phase Analysis Results
Figure 6 presents the X-ray diffraction patterns of different groups.All peaks align with the characteristics of the CL group.The results indicate that femtosecond laser and femtosecond laser combined HA crystals do not induce phase transformation.

Flexural Strength Test Results
The average flexural strength of each group is presented in Table 3.The FHA group exhibited the highest strength, followed by the FL group, while the CL group showed the lowest flexural strength.However, there was no significant difference observed among the three groups.

Biological Characterization Results
It's evident that rough surfaces of the FS and FHA groups resulted in pronounced changes in cell morphology.In the CL group (Figure 7A), merely a small number of round and wrinkled cells were observed attached to the surface.However, in the FL and FHA groups (Figure 7B,C), a large number of cells adhered to the grooves and flat spaces between them.The cells were aligned parallel to the grooves.Importantly, the FHA group exhibited significantly higher cell adhesion compared to the CL and FS groups.

Discussion
The surface properties of dental implants have been recognized as crucial factors influencing early osseointegration, which include surface topography [24] and chemical composition. [25]Zirconia dental implants often exhibit low surface bioactivity, leading to inadequate osseointegration and limiting their clinical application.In order to enhance the surface bioactivity of zirconia dental implants, a combination of micro-groove design and calcium phosphate deposition was implemented on the zirconia surfaces in this study.
The fabrication of the micropatterns through femtosecond laser treatment enabled high precision and reproducibility.The formation of regular micro-grooves was observed, evenly distributed, and aligned parallel to the laser processing direction.The groove edges appeared smooth without any micro-cracks or fissures on the surface, consistent with our previous work. [11]n this study, the surface roughness of the FS group and FHA group is significantly greater than that of the CL group, indicating that the femtosecond laser treatment increased the surface roughness of zirconia.However, it is demonstrated that if the sample is hydrophilic, an increase in surface roughness enhances its hydrophilicity.Conversely, for hydrophobic samples,  an increase in surface roughness results in an enhancement of their hydrophobicity, [26] which is consistent with our study since zirconia is hydrophobicity.
The interactions between cells and the various kinds of controlled microtopographies on implant surfaces have increasingly become the main focus in the development of dental implants. [18]lthough micropatterns can enhance osteogenic differentiation of BMSCs, there is no consensus on the optimal scale and the mechanism is still unknown.Microgrooves with a width ranging from 4 to 60 μm and ridge width less than 10 μm have the potential to induce larger focal adhesions (FAs), wellorganized cytoskeletons, and enhanced cell spreading areas of BMSCs.This, in turn, promotes osteogenic differentiation. [27]hao et al. [28] demonstrated that grooves with widths equal to or smaller than the cell size promote osteogenic differentiation.In this study, the width of microgrooves in FS and FHA groups was ≈15 μm, which is within the optimal scale.BM-SCs were spindle-shaped with the cytoskeleton stretched along the grooves.This indicates that surface topography induced by femtosecond laser has contact guidance and influence on cellular functions.However, there was no statistically significant difference in cell proliferation among the groups.This could be attributed to the relatively wide spacing between the microgrooves.In future studies, we will reduce the distance between the micro-grooves to investigate its potential impact on cell proliferation.
When the specimen surface was irradiated by the laser, twophoton absorption occurred, transferring the energy of photons to the electrons causing a sharp rise in electrons temperature. [29]he high-temperature plasma resulted in a significant increase in solution temperature and vaporization, leading to the rapid accumulation of calcium and phosphate ions in the surrounding area, achieving a high degree of supersaturation. [30]Subsequently, solid precipitation occurred in the supersaturated solution, resulting in the formation of insoluble calcium phosphate.In the FHA group, dark-colored granular deposits were uniformly distributed along the lateral wall and bottom of the micro-grooves.This finding is consistent with the Ca/P ratio of 1.5 observed in the deposits from the FHA group.To further explore the specific composition of these deposits, three groups of zirconia samples and hydroxyapatite power were analyzed using FTIR.When comparing with the CL and FS groups, it can be observed that the peaks ≈1090 and 1032 cm −1 appeared in the FHA group, overlapping with the peaks of PO 4 3− in HA, indicating the presence of phosphate on the surface of the FHA group.However, unlike the spectra of HA, there was no peak corresponding to OH − in the FHA group.The absence of an OH − peak in the FHA group suggests the dehydration of HA crystals, resulting in the formation of CP.The chemical composition of CP is similar to that of bone mineral matrix, exhibiting certain biological activity and the ability to stimulate bone regeneration. [31]tudies have demonstrated that CP can induce the expression of osteogenic genes and significantly enhance the activity of alkaline phosphatase, thus promoting osseointegration. [32,33]Rameshwar et.al.reported that the nanocomposite composed of CP nanoparticles and graphene oxide could accelerate the differentiation of stem cells into osteoblasts. [34]In this study, the FHA group exhibited more cell number and higher cell adhesion compared to the CL and FS groups, which is consistent with the previous study.It is indicated that the production of CP in the microgrooves had a synergism function to the rough surface contact guidance.
The XRD analysis indicated that femtosecond laser ablation did not induce any phase transition of material, consistent with the previous findings. [14]However, no corresponding peak was observed for the deposition of calcium phosphate in the microgrooves, which could be attributed to its low content.It is important to note that XRD may not detect the presence of a substance when its content is less than 5% of the total content within the tested area.
Mechanical properties play a crucial role in dental implants.In this study, although no statistical difference was observed among the three groups, the flexural strength of zirconia treated with femtosecond laser (FS and FHA groups) exhibited a slight increase compared to the control group.The slight improvement in mechanical properties of zirconia may be attributed to reduced crack damage caused by external conditions, rather than phase change toughening of zirconia. [35]

Conclusion
The fabrication of microstructures with calcium phosphate (CP) deposition on zirconia surfaces was successfully achieved using a combination of femtosecond laser and hydroxyapatite (HA) crystals.These modifications without any surface damage did not affect the crystal structure or the flexural strength of the zirconia material.The optimized rough and bioactive surface could guide BMSCs to align to the microgrooves and stretch the cytoskeletons.Further in vitro and in vitro investigations will focus on evaluating the optimal width and depth of microgrooves on osteogenic differentiation of BMSCs and the molecular mechanism.

Figure 1 .
Figure 1.Schematic diagram of femtosecond laser treatment combined with HA crystals.

Figure 2 .
Figure 2. SEM micrographs of zirconia with different surface treatments.A,B) presented the smooth surface of CL group, C,D) presented the rough surface of the FS group, and E,F) presented modified surface of the FHA group.(G) is the magnification of the red rectangle in (F).

Figure 3 .
Figure 3. EDS spectra of specimens in A) CL, B) FS, and C) FHA groups.D) presents the elements analysis of the area between micro-grooves in the FHA group.

Figure 4 .
Figure 4. Laser confocal microscope 3D micromorphology images of zirconia surfaces in different groups.

Figure 5 .
Figure 5. FTIR spectra of specimen in CL, FS, and FHA groups.HA Powder spectrum was used as a reference.

Figure 6 .
Figure 6.X-ray diffraction spectra with different surface treatments.

Figure 7 .
Figure 7. SEM images of BMSCs on the different surfaces after 1 day of incubation.A) is CL group, B) is the FS group, and C) is the FHA group.

Table 1 .
Surface roughness values of the three groups (Mean ± Standard deviation).

Table 2 .
Contact angle of the three groups (Mean ± Standard deviation).
a-b) Same superscript letters represent significant no differences for inter-group comparisons (p >