Investigation of the Biocompatibility of Laser Treated 316L Stainless Steel Materials

Abstract

In this study, 316L stainless steel materials, which are widely used in the industry, were produced by investment casting management. Depending on the microstructure, the hardness values constitute an important stage of the properties that can be developed and controlled. For this purpose, the differences between the microstructure and hardness properties of 316L stainless steel, which is produced by the investment casting method, and 316L stainless steel, which is currently used commercially, were examined. The changes caused by the fiber laser on the surface of 316L materials produced with two different production methods were examined. It was observed that the laser used made different changes in the surface structure of the 316L material produced by both methods. Since the surface of the material is a buffer between body fluids and biomaterial, it is known that there is a relationship between surface properties and biocompatibility. In this study, the L929 cell growth test, one of the cytotoxicity tests, was applied and thus, how laser surface treatment affects the biocompatibility of 316L materials produced by both methods was comparatively examined.

1. Introduction

The presence of nickel in concentrations higher than 8%, in stainless steels, is what stabilizes the austenite phase at room temperature; these stainless steels are referred to as austenitic stainless steels [1]. Stainless steels are divided into five groups based on their microstructure. These steels have high mechanical qualities and are resistant to corrosion; as a result, they find use in many different industries, including petrochemistry, treatment facilities, and the medical industry [2,3]. In orthopedic research, austenitic stainless steels are the alloys that are utilized the majority of the time. This industry makes extensive use of alloys of the 316L type, which may acquire a high degree of hardness by very simple deformation. Because it is not magnetic, the austenitic phase offers advantages in terms of corrosion resistance in ferrous alloys that are superior to those offered by other phases. According to the research that has been conducted, the pitting corrosion resistance of an alloy can be improved by the addition of a trace quantity of molybdenum [4,5].

Up until the Second World War, the investment casting technique was only used commercially by jewelers and dentists. After the war, however, the method began to find widespread use in industry, particularly for the production of investment components in the aircraft, textile, electronic, and machinery sectors of the economy. In this technique, two distinct approaches are utilized, namely graded investment casting and ceramic shell investment casting [6]. Both of these approaches are described in greater detail below. During the process of investment casting, metal molds representing the prototype of the item that will be manufactured are created. After the wax is injected into the metal mold to create the models, the wax models are joined so that they are all on the same runner to create a model tree in the shape of a cluster. This enables the mass production of several parts at the same time, which allows for greater efficiency. The refractory mud has been used to coat the model tree that was made. This coating may come in the form of shells or it may be manufactured in grades. Both options are possible. Following the application of the coating, the cavity of the mold can be acquired by melting the wax model material, which is then removed from the mold once it has been heated. After the wax that was deposited on the newly created ceramic shell has been removed, the shell of ceramic is next burned. After the metal has been heated to a temperature higher than its melting point and melted, the casting process involves pouring the molten metal into the cavities that have been made in order to create the desired shape. Following the process of casting, the final product is obtained by the subsequent steps of shell breaking, cutting, and surface treatments [7,8].

Applications for micro and nanostructures generated by lasers on metal or semiconductor surfaces can be found in a wide variety of disciplines, ranging from manufacturing to medicine [9]. When compared to other methods, laser treatment of implant surfaces allows for the best possible surface topography, the lowest possible pollution levels, and the creation of complex geometric shapes. These findings are based on the findings of studies that have been conducted over the past ten years [10,11]. When the laser beam is focused on the substance that is being targeted, some of the laser beam is absorbed by the material, while the remaining portion is reflected by the material. The energy that is received by the surface of the target material causes the surface of the target to begin to heat up. During the laser heating process, the target material might not undergo phase shift and might continue to be solid in some circumstances, depending on the laser power per unit area. In the event that the intensity of the laser radiation is increased, the material’s surface temperature will rapidly rise to the point where it will melt as a consequence of the heating process that is caused by the material’s absorption of high-intensity laser radiation. This will cause the material to melt. Surface evaporation takes place whenever sustained laser treatment is performed on the surface of the molten material [12,13,14]. Figure 1 shows schematically the physical processes that take place [11]. When the laser power is close to the damage threshold of a surface, the interaction of the laser beam with the surface generates nano-scale periodic structures/ripples. The nanoscale periodic ripple formation has been commonly attributed to interference between the incident laser beam and the surface-scattered wave. Micro and nano structures created with lasers also affect the wettability properties and cell adhesion ability of a surface [15].

In surface modification applications involving lasers, one of the fundamental principles is causing abrasion on the material surface in order to modify it. This is accomplished by concentrating a laser pulse of high intensity on the surface of the target material for a brief period of time [16]. The main applications that include the laser etching process, which occurs as a result of laser-material interaction, are as follows: laser material processing applications, including laser drilling, cutting, welding [17,18]; laser micro-machining applications, including the production of structures such as medical devices and materials, microsensors, microelectronics [19]; chemical analysis, including laser-induced plasma spectroscopy applications [20], nano-structure fabrication [21], and film storage [22,23]. Laser applications are fascinating because of the benefits that lasers offer over more conventional methods. For instance, mechanical etching methods have been replaced with laser micromachining in materials that are difficult to etch using standard mechanical methods [24,25]. Laser processing is a thermal process, and the efficiency of the process is dependent not on the mechanical qualities of the material to be processed, but rather on the thermal and optical properties of the material to be treated by the laser. When compared to other processing methods, laser processing offers a number of benefits in a material that is either extremely brittle or extremely hard [26]. The significant benefit of the laser’s capacity to process at a fast speed and without contact is a significant advantage that makes it superior to a wide variety of conventional production processes [27]. Because the transmission of energy between the laser and the material is accomplished through radiation, the material is not harmed in any way by the mechanical impacts of the process. It is advantageous to employ lasers in the production of small parts in the biomedical, aerospace, electronic, and manufacturing industries because of the fact that lasers provide processing that is quick, clean, precise, and non-contact in addition to being effective. A schematic representation of the laser material processing application can be found in Figure 2 [11].

In order for a biomaterial to be produced by the investment casting technique to be used in the living body, it must meet some biocompatibility requirements such as strength, corrosion resistance, non-toxicity, non-allergic, and non-immunological [28]. The biocompatibility of a material is determined by in vitro and in vivo tests involving the interaction of the material with biological fluids and cells. The cytotoxicity test is the first biocompatibility test to be performed before beginning detailed in vivo testing in laboratory animals. To minimize the unnecessary use of test animals, ISO 10993-2 recommends in vitro testing of medical device materials before starting animal testing [29]. This in vitro test is easy to perform and results are available in 2–3 days. The cell lines most commonly used in this assay are L929, 3T3, or V79. In the L929 Cell growth test, cell cultures are incubated with test product extracts at 37 °C for 2 days in a humidified CO₂ incubator. The negative control can be high density polyethylene (HDPE) and the positive controls can be cadmium chloride, PVC (polyvinyl chloride)-stabilized Organotin (PVC-Sn). Tri-cultures are preferred in each group. At the end of 24 and 48 h, cytotoxicity is recorded based on the morphology of the cells and the area of growth inhibition. If 70% or more of the cell layers contain rolled or fragmented cells, the assay is classified as “failed”. However, if there are more cells that show morphologies that adhere to the surface by forming extensions, the result is considered successful [30,31].

In this study, 316L stainless steel materials were produced as experimental samples by the precision casting method. For the purpose of this investigation, experimental samples of 316L stainless steel materials were created using the precision casting process. After undergoing laser processing, the biocompatibility of 316L alloys that are currently on the market was studied for the purpose of this study. These alloys were created by following the investment casting method in its entirety from start to finish.

2. Materials and Methods

2.1. Material Production and Preparation

Experimental material production was carried out using investment centrifugal casting device. Production of wax models, coating with refractory material, melting, and unwinding of wax, baking, casting, mold-breaking, and grinding operations were performed. The specimens were produced with a diameter of 35 mm and a height of 50 mm. The chemical compositions of the 316L materials fabricated and commercially supplied in the experimental runs are given in

Table 1. Chemical composition of 316L material used in experiments.

	316L Investment Casting (wt%)	316L Commercial (wt%)
C	0.02	0.02
Si	0.80	0.69
Cr	16.60	16.94
Mo	2.45	2.28
Mn	0.86	0.97
Ni	11.75	10.91
Al	0.028	0.024
Cu	0.4	0.39
V	0.057	0.06
Fe	Other Part	Other Part

.

After the procedures of cutting and molding the manufactured samples, as well as the samples that were commercially available, the surfaces of the samples were sanded with wet sandpaper with a mesh size of 200, 400, 600, 800, 1000, and 1200, respectively, until the surface roughness was erased. These surfaces were given a polish with diamond paste with a 6 micron grain size, and then they were prepared for etching. Electrolytic etching was performed using a solution containing 10% oxalic acid, with a voltage of 12V and a current intensity of 2A, in order to show the microstructure image of 316L materials. Examinations of the microstructure were carried out using an optical microscope of the brand Nikon ECLIPSE L150, with magnification ranging from ×50 to ×1000. Image analysis software of the Clemex VisionLite brand was utilized in order to carry out measurements of grain size. The hardness of the test samples was determined using a device of the brand MikroBul 1000D, which measures hardness. Hardness measurements were carried out by applying an HV1 (1000 g) load ±3 standard deviations. Hardness values were determined by taking the average of 10 hardness measurements from each sample.

2.2. Laser Processing

For surface modification, nano pulsed 1064 nm wavelength Nd:Yag Fiber laser in Kocaeli University Laser Technologies Research and Application Center (LATARUM) and a galvo-laser head (scanner head) suitable for this laser were used. The experimental parameters used are given in

Table 2. Laser system parameters used in experimental work.

Laser Type	Fiber
Power	14 W
Pulse Duration	<50 ns
Frequency	20 kHz
Gas Type	Argon (02–05 bar)
Line Space	100 μm
Velocity	10 mm/s
Beam Diameter	<50 micron
Structured Area	10 × 10
Time	123 s

.

2.3. Biocompatibility Tests

WST-1 (Roche) viability test was performed to determine whether metal plates had a cytotoxic effect on L929 cells or whether cells could adhere to these scaffolds and proliferate. Cells were removed from the flask in which they were grown using 0.05% Trypsin solution. For this test, surfaces with an area of 1 cm² were sterilized in 70% ethanol for 30 min in an ultrasonic bath. In the Thermo Scientific S2020 brand biosafety cabinet, samples were placed in 24-well plates and $2.5\times {10}^4$ cells per cm² were seeded. Cell-inoculated samples were cultured in High glucose-DMEM medium containing 10% FBS and 1% Pen/Strep at 37 °C in a N-Biotek NB203-XL brand incubator with 8% CO₂. On the 1st and 3rd days, the WST-1 test was performed to determine the normal cell culture polystyrene (positive control) and the viability rates depending on the biochemical activities of the cells on the samples. In order to determine the cell viability of cells cultured, polystyrene and cells seeded in the samples on the 1st day, firstly, a 5% WST-1 solution was prepared in the dark with L-DMEM medium containing 10% FBS and 1% Pen/Strep. Then, 1000 μL/well of the prepared solution was placed in 24-well plates, and cell samples were quickly transferred into the solution. Three of each sample were studied. The plate, which was covered with aluminum foil to avoid light, was kept at 37 °C for 1 h in an incubator with 5% CO₂. In a 96-well plate, 200 μL of each sample’s medium was taken from each well and 3 wells were filled. Then, absorbance values were read at 440 nm wavelength using a microplate reader (FlexStation3, Molecular Devices). On the 3rd day, studies were carried out in the same way and absorbance values were recorded.

3. Experimental Results and Discussion

3.1. Microstructure

The microstructure photograph extracted from the 316L stainless steel sample that was manufactured using the investment casting process is presented in Figure 3a. When the picture of the microstructure is examined, one notices that the structure is made up of a coarse-grained austenite matrix. In their investigation, Emre et al. demonstrated comparable results in the microstructure photographs of 316L stainless steel base material [32].

Figure 3b gives a picture of the microstructure from a commercially available 316L stainless steel sample. Looking at the microstructure picture, it is seen that the structure consists of a fine-grained austenite matrix structure. The reason why it is fine-grained compared to the investment casting method is due to the fact that these samples were produced by rolling. Grain structures are observed depending on the crystalline structure in all metals produced by the Casting Method. Since the Investment Casting method is also a casting method, it is also reported in the literature that the grain structure is observed as a result of the metal passing from the liquid state to the solid state [33].

3.2. Hardness Test Results

The results of testing the hardness of 316L stainless steel and samples of commercially available 316L stainless steel manufactured by investment casting are displayed in Figure 4. The hardness value for investment casting and commercial 316L stainless steel material was measured as 132 HV and 173 HV1, respectively. Student’s t-test was applied for hardness values and it was found as t = −1. It is seen that the hardness values of 316L stainless steel materials commercially available in the literature are high. This is thought to be due to the higher average grain size values of the samples fabricated by the investment casting method. It has also been stated in the studies in the literature that the 316L stainless steel materials produced by the casting method are coarse grained. The parts fabricated by the investment casting method have a coarse-grained structure as in the general casting methods due to slow cooling.

3.3. Surface Modification

The surface images obtained after laser surface treatment using the parameters given in Table 2 are shown in Figure 5.

Laser lines caused by laser scanning were clearly visible in the commercially produced 316L material as a result of the surface treatment with laser, whereas these lines were less visible or partially disappeared in the investment cast sample. The difference in microstructure of both samples and the resulting surface tensions can explain this situation. Because of the thermal effects that occur during laser processing on large-grained surfaces, the microstructure changes in the areas close to the surface, causing the lines to deteriorate and partially disappear. Since coarse grained structures have a lower ablation threshold, it is thought that the coarse grained material fabricated by investment casting is more damaged by the laser power and therefore the laser lines are lost. However, since the biocompatibility will be examined, the laser parameter was not changed according to the ablation threshold.

3.4. Biocompatibility Test Results

The biocompatibility of 316L surfaces was investigated by in vitro cell culture tests. Cell culture studies were carried out using the L929 cell line obtained from mouse connective tissue fibroblast cells. The obtained scanning electron microscope images showed that the cells adhered much better to the 316L investment cast samples and the cells could spread on the surface by forming extensions such as filopodia and lamellipodia (Figure 6). On the other hand, cell adhesion on 316L commercial samples showed numerically similar properties, but when the cell morphology is examined, it is seen that the extensions formed are not as in the investment cast sample (Figure 7). In addition, at the end of the first day in Figure 8, it is seen that the third day numbers of the cells that adhered better in the samples produced by the investment casting method increased significantly. Cell morphologies were observed in the laser-processed samples by forming extensions on the first day, and it was determined that the cells proliferated on the third day, as shown in Figure 8 and Figure 9.

In cytotoxicity studies, the ability of L929 cells to adhere to the surface of metal plates was investigated and numerical data were obtained. During the study, tests were carried out with four sample groups, 316L commercial, 316L investment casting, 316L commercial laser, 316L investment casting laser, and tissue culture polystyrene (TCP) group for positive control. Three replications were made for each prepared group. At the end of the WST-1 test, the number of viable cells at the end of 24 and 72 h were determined (Figure 10 and Figure 11). In the literature, it has been stated that metabolic activities of the cells, whilst cell morphology is seen on different material surfaces [34], and also material surfaces such as stainless steel and phase morphologies are also important [35,36,37,38].

As seen in Figure 10, the highest cell number was counted in the 316L investment casting sample after 72 h. The number of cells attached to the commercial 316L sample metal surface after 72 h is very close to the number of cells on the TCP, but lower than the 316L investment casting. In the microstructure, the difference in hardness and the differences in surface tensions are evaluated, and it is thought that 316L commercial samples with small grain size and high hardness value have a negative effect on the number of cells due to the high surface tension. The lowest cell count was detected in the laser treated 316L investment casting sample after 24 h. The reason for this is that the investment cast samples have less prominent lines than the 316L commercial sample, considering the metal surface topography after laser treatment (Figure 5), and it is thought that the adhesion and growth of small cells in these irregular lines is insufficient. Similar results are also seen in the study of Köse et al. [39]. A significant increase in cell numbers was observed with the increase in duration from 24 h to 72 h. This shows that cells at 72 h, which show growth compared to 24-h cells, can adhere to metal surfaces more easily, as seen in Figure 10 and Figure 11.

4. Conclusions

In this study, precision casting of 316L stainless steel material was performed. Depending on the production method, the effect of fiber laser on the biocompatibility of materials was investigated. The results obtained are summarized below:

1.

It was determined that 316L stainless steel materials can be produced with the desired properties with the investment casting method.

2.

The grain structure determined by commercial rolling, for example, was found to be coarser than the other methods as a result of microstructure investigations applied to 316L stainless steel materials. This is because the materials produced by the casting process have a large grain size.

3.

The hardness test results applied to 316L stainless materials produced by casting method and commercially available were measured as 132 HV1 and 173 HV1, respectively. The low hardness values are related to the coarse grained structure as a result of the casting process.

4.

Using a fiber laser, desired patterns can be created on 316L material.

5.

The fiber laser has different effects on both material surfaces, which is explained by the difference in microstructure.

6.

It has been observed that the lines created by laser in the samples with coarse grain structure and low hardness value produced by investment casting are wider than the commercial 316L sample due to surface shrinkage.

7.

Investigations made as a result of Cell Tests showed similar cell growth values for the surface morphology of 316L stainless steels, which varies depending on the production method.

8.

The highest cell count was counted in the 316L investment casting sample after 72 h. It has been determined that materials with high biocompatibility can be produced by the investment casting method compared to commercial 316L metal materials.

9.

The 316L cell growth assays produced by Investment Casting and laser processed showed the best morphology on sem images, although it showed the least number on day one. At the end of the third day, the cell numbers of the laser-processed samples produced by precision casting and the commercially obtained and laser-processed samples became closer to each other.