The influence of laser frequency and groove distance on cell adhesion, cell viability, and antibacterial characteristics of Ti-6Al-4V dental implants treated by modern fiber engraving laser

Abstract

Objective

Micro-nano scale surface modification of Ti-6Al-4V was investigated through the fascinated modern fiber engraving laser method. The process was performed at a high laser speed of 2000 mm/s, under different laser frequencies (20–160 kHz) and groove distances (0.5–50 μm).

Methods

Topographic evaluations such as Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FE-SEM) were used to identify the quality and regularity of patterns. The proliferation of human osteoblast-like osteosarcoma cells (MG63) was analyzed by MTT assay for up to 72 h. Also, the plate counting method was used to quantify the viability potential of the modified surface against Escherichia coli bacteria.

Results

The cellular viability of the sample modified at the laser frequency of 20 kHz and grooving distance of 50 μm increased up to 35 and 10% compared to the non-treated and control samples, respectively. In the case of the surface modification at lower grooving distances range between 0.5–50 μm, the maximum laser frequency (160 kHz) applied leads to lower pulse’s energies and less bacterial adhesion. Otherwise, at groove distances more than 50 μm, the minimum laser frequency (20 kHz) applied reduces the laser pulse overlaps, increases the cell adhesion and antibacterial properties.

Significance

Surface modification by the fiber engraving laser process significantly enhances the cell adhesion on the surface. As a result of such roughness and cell adhesion enhancement, the surface toxicity feature diminished, and its antibacterial properties improved.

Introduction

Ti-6Al-4V is one of the most significant biomaterials utilized in medical prostheses and dental implants because of its superior physical and mechanical properties such as high strength to weight ratio, high fatigue strength, good ductility, machining capability, excellent corrosion resistance, high-temperature stability and especially great biocompatibility with body’s tissues [ , ]. The adhesion of bone cells to the implant surface is highly dependent on the surface roughness and its energy. Especially in the case of titanium alloys, the neutral bioavailability of this material limits bone cell adhesion on the surface [ , ]. Implant’s surface roughening is one of the most effective ways to enhance the cell-implant adhesion as well as the cell proliferation rate [ ]. While the macro-scale surface roughness effects on the long-term mechanical stability of implants [ ], micro-scale roughness enhance the bone generation and its torque resistance in the body [ , , ], and the nanoscale one plays a vital role in protein absorption and cell-implant adhesion [ ]. Furthermore, the wettability enhancement as a result of surface roughening can significantly improve the protein absorption, cell adhesion, and, subsequently, the cell proliferation rate [ ].

Surface roughening of medical implants can be performed by a variety of methods like chemical etching, anodic oxidation, plasma spraying, additive manufacturing, lithography, coating and laser engraving [ ]. The use of lasers is accepted as the most versatile and cost-effective method instead of traditional surface texturing methods that made transcendent strides in recent years [ ]. In this method, the laser is focused and aligned by the light elements and creates a small effect point with a high energy density on the surface. The laser beam has a high temperature that causes melting, ablation, and rapid partial evaporation and subsequently creates microcavities and roughens the surface [ , ]. Different kinds of lasers have been evaluated so far for the surface roughening of medical implants. Trtica et al. [ ] improved the titanium implant’s biocompatibility, through its surface roughening and oxidation using Nd: YAG laser. UV laser modification of the Ti-6Al-4V implant has simultaneously created micro-grooves and tiny pores that can provide proper locations for the growth of bone cells [ ]. Surface modification of titanium alloy with a CO 2 laser also recognized for improving the cell adhesion and its proliferation rate effectively [ ]. As well, through surface engraving with a lithographic laser, it was indicated that different groove patterns could affect the growth of bone cells on the titanium implants [ , ]. In our recent paper, it was also indicated that morphology and adhesion of cells on the surface of Ti-6Al-4V alloy could be enhanced through the optimization of Nd: YAG laser frequency [ ].

In this paper, the fiber engraving laser unique method is introduced as a fast and facile process for surface modification of Ti-6Al-4V alloys, which never has been used for metal’s surface modification compared to previous processes. This delicate and clean method enables engraving the variety of patterns on implant’s surface, with high accuracy and various adjustable parameters including higher frequencies [ ]. Although this method is generally utilized for improving the mechanical properties of some alloys, its effect on the biological properties enhancement of implants studied just in a few papers [ , ]. Chan et al. [ ] modified the surface of Ti (Grade2), Ti-6Al-4V, and Co-Cr-Mo (Grade5) alloy with 1064 nm fiber engraving laser in a nitrogen atmosphere. Laser modification diminished the hydrophobicity and generated the thick nitride-oxide layers on the modified surfaces, which reduced the antibacterial properties. It is also reported that increasing the laser energy density of the fiber engraving laser process raises the amount of oxide compounds on the surface, which make the titanium alloy suitable for dental implant applications [ ].

This paper aims to investigate the fiber engraving laser process for surface modification of Ti-6Al-4V implants and biological properties relationship. Laser frequency and grooving distance parameters were recognized as the most important variables of fiber engraving laser process, which have complicated effects on the surface roughness and biological properties of modified surfaces. The structural and topographic investigation of the modified samples also revealed that the fiber engraving laser method has a great potential for the creation of micro to nano scale roughness on the surfaces and the interior of the samples contained the original microstructure and did not change to the martensitic zone, which is considerably important for implants’ stability in body [ ]. Also, the effects of fiber engraving laser parameters were evaluated for the first time on the cell adhesion, toxicity, and antibacterial properties simultaneously to find the best optimum modified sample. Engineering optimization of process parameters results in the modification of surfaces with desire wettability and enhancement of cell viability. Because of such unique features, the fiber engraving laser process seems to be a worthy alternative to CO 2 and Nd: YAG lasers in medical equipment.

Experimental procedures

Surface modification process

Ti-6Al-4V alloy sheets (90.692 Ti, 5.757 Al, 3.542 V, 0.273 Fe, 0.051 SO 3 ) were sanded to remove surface oxides and ultrasonically cleaned in ethanol, acetone and distilled water. Surface modification was performed by fiber engraving laser device at a high laser speed of 2000 mm/s, an average power of 80 W, and in-room atmosphere. The process was done with a single mark loop at different laser frequencies of 20, 80, and 160 kHz and various grooving distances of 0.5, 1, 4, 10, 20, 50, and 100 μm.

Metallurgical characterizations

The roughness profiles of different modified surfaces were measured with a surface roughness tester (model surtronic 25). Roughness in the vertical direction is the best component for comparing different samples, and in horizontal direction is not comparable for different samples, because firstly the measuring device is not capable and accurate enough, secondly the roughness checker’s probe scanned area is not the same for different samples, thirdly the roughness in that zone is much less than vertical roughness and therefore cannot indicate surface roughness. Subsequently, roughness arithmetic mean deviation (R a ), sharpness or widths of peaks and valleys (R ku ), maximum profile peak height (R p ), and minimum profile peak height (R v ) parameters were extracted from the roughness profiles using TalyProfile Lite 7.1 software. Three-dimensional surface topography of modified samples was investigated by the Atomic Force Microscopy (AFM, model Veeco CPII). The wettability analyses were performed according to ASTM D7490-13 standard, while the contact angles were photographed by optical microscopy (OM, model Dino-Lite Digital Microscope). As well, surface topography and cross-sectional microstructure were characterized by a Field Emission Scanning Electron Microscopy (FE-SEM, model FEI Nova NanoSEM 450) at 10 kV, equipped with an Energy Dispersive X-Ray Spectroscopy (EDS, model TESCAN). Eventually, surface composition and its crystallographic structure were analyzed using X-ray diffraction method (XRD, model X’Pert-MPD).

In vitro cellular evaluation

Cell culture

Human osteoblast-like osteosarcoma cells (MG-63) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), and penicillin/streptomycin at 37 °C, 5% CO 2 and 90% humidity. The medium was changed every 2–3 days.

Cell viability assay

Ti-6Al-4V samples with the size of 6 × 6 × 2 mm 3 were sterilized by autoclaving at 121 °C for 30 min, the MG-63 cells were seeded on samples to study the cell proliferation and in-Vitro cytotoxicity of the samples by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. MG-63 cells produce mitochondrial lactate dehydrogenase enzyme, which reduces MTT salt to insoluble formazan crystals. The experiments were performed with 5 × 10 3 cells for 12, 24, 48, and 72 h in triplicates for each sample.

Afterward, the culture medium was removed, and MTT salt solution (200 μl, 0.5 mg/mL) was added into each well and placed in an incubator for 4 h. Then the solution was removed from the cells, and 200 μl of DMSO was added to dissolve the purple formazan crystals. Finally, 100 μl of the purple solution was transferred to 96 well plates, and the absorbance of each well was evaluated at the wavelength of 570 nm using a microplate reader (μQuant BioTek).

Cell adhesion assay

After 48 h of culture, MG-63 cells on Ti-6Al-4V samples were fixed using 2% formalin and 2.5% glutaraldehyde in PBS, pH 7.4 for 15 min, to complete fixation samples kept in the fridge for 2 h and subsequently rinse with water. The samples were dehydrated using a graded series of ethanol (50%, 60%, 70%, 80%, 90% and 100%). Afterwards, dried samples were mounted on the copper stubs using adhesive carbon tapes and gold was sputtered on the samples to form a thin (∼20 nm) layer on the surface. The SEM images were captured at 15 kV accelerating voltage using FE-SEM.

Bacterial analysis

The plate counting method was used to quantify the bacterial adhesion property of samples. For such analysis, E. coli ATCC25922 bacterial cells were cultured overnight at 37 °C in Luria–Bertani (LB) broth medium in a continuously shaking incubator. Sterilized samples were horizontally placed into 12 well plates, and 50 μL bacterial solution (∼10 8 CFU/mL) was dropped on each sample. The well plates were incubated at 37 °C for 3 and 6 h, then the bacterial solution was removed from the cultured sample, and their surfaces were washed using sterile phosphate buffer. After that, each sample was immersed in 1 mL of culture medium and soaked for one minute until the adherent bacteria were suspended. Finally, the suspended bacteria were diluted by serial dilution and cultured on agar plates for 16 h to count the colonies number.

Statistical study

Statistical analysis was assessed by using a GraphPad Prism 8 statistical software package. The quantitative results were presented as the means ± SE for each group, the differences results were compared using the ANOVA two-way analysis of variance (ANOVA) for multiple comparisons. A p value of <0.05 was considered statistically significant.

Results and discussion

Surface topography evaluation as a function of grooving distance

Fig. 1 a shows the schematic of the fiber engraving laser technique. Through this process, a laser beam aligned by the light elements and focused on the small point of the surface. The high amount of absorbed laser energy pulses in nanosecond (ns) regime, causes consequent remelting and partial evaporation. Considerable amount of material was not vaporized but only melted and solidified rapidly [ ]. Programming the laser travel path makes it possible to groove the surface with specific patterns, like that shown in the inset of Fig. 1 a. Here, the effects of such inter-grooves distances were investigated on the surface topography of Ti-6Al-4V implants. Fig. 1 b shows the roughness profiles of the samples modified at different grooving distances. As can be seen, the average roughness of all modified samples is between 1.1 μm–3.7 μm which is much higher than the untreated sample (≈0.45 μm). As well, much rougher surfaces are distinguished for the samples modified with lower grooving distance less than 10 μm, where declined widths of the valleys and peaks resulted in the creation of compact roughness profiles. Fig. 1 c plotted the variation of different roughness parameters by grooving distance. While R a , R p, and R v parameters exponentially decreased at higher distances, R ku represented a gradually rising trend High groove distances leads to the reduced number of pulses per unit area as well as lower pulses interactions with each other. In this regard, lower induced surface energy was found to results in a decreased roughness peak heights (R p and R v ) and average surface roughness (R a ). Meanwhile, due to the creation of roughness peaks between grooves ( Fig. 1 a), increasing the grooving distances is proportional with widening the roughness peaks and so increasing the R ku parameter.

Fig. 1
(a) Schematic of the surface modification process by fiber engraving laser, (b) Roughness profiles of untreated and modified surfaces at the constant frequency of 20 kHz and different grooving distances, (c) Surface roughness variations as a function of grooving distance.

Accordingly, the grooving distances higher than 4 μm result in surface roughness of 1–3 μm, the ranges that desired biological properties of implants are also expected.

Fig. 2 investigates the metallurgical characteristics of the samples modified by the fiber engraving laser process at different grooving distances. Engraving of all the samples with regular patterns of parallel rows is evident in the low magnification FE-SEM micrographs of Fig. 2 a. Higher magnification FE-SEM micrographs ( Fig. 2 b) also reveals lots of spherical grains and cavities around the grooves, originated by subsequent remelting and rapid solidification. In the case of sample modified with 0.5 μm grooving distance, micro and macro cavities are distinguished between the solidified spherical grains. While macro cavities are such a desired sticking area for ≈20 μm bone cells [ ], microcavities can absorb the smaller bacteria of ≈2 μm and reduce the antibacterial properties. Accumulation of fast solidified spherical grains on top of each other and also inter-grains cavities are much less common in the samples modified with higher grooving distances, instead molten sputtering phenomenon can clearly distinguish at such modified surfaces. In this regard, increasing the grooving distance is accompanied by lower laser pulses interactions and also a lower cooling rate, allowing the partially melted alloys to be spread over the surface [ ]. The cross-sectional FE-SEM images of Fig. 2 c suggests that increasing the grooving distances from 0.5 to 50 μm, accompanied by reducing the thickness of the modified layer from 28.98 to 0.78 μm, probably due to the lower energy induced in the surface as well as lower laser pulses interactions. According to the AFM images of Fig. 2 d, the fiber engraving laser method is also capable of modifying the surface with nanoscale roughness, an advantageous feature which results in improving the cells adhesion, accelerating the implant-bone bonding, and enhancing the antibacterial properties [ , ].

Mar 21, 2021 | Posted by in Dental Materials | Comments Off on The influence of laser frequency and groove distance on cell adhesion, cell viability, and antibacterial characteristics of Ti-6Al-4V dental implants treated by modern fiber engraving laser
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