Article 1

Effect of High-Pressure Torsion on Mechanical and Functional 2

Characteristics of 316LVM Austenitic Steel 3

Olga Rybalchenko 1, Natalia Anisimova 1,2,3*, Natalia Martynenko 1, Georgy Rybalchenko 4, Alexey Tokar 1,3, Elena 4
Lukyanova 1, Dmitry Prosvirnin1, Mikhail Gorshenkov 2, Mikhail Kiselevskiy 2,3 and Sergey Dobatkin 1* 5

1 A.A. Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences, 119334 Mos- 6
cow, Russia; rybalch@mail.ru (O.R.); nmartynenko@imet.ac.ru (N.M.); helenelukyanova@gmail.com (E.L.); 7
dprosvirnin@imet.ac.ru (D.P.); tokarb2005@mail.ru (A.T.) 8
2 N.N. Blokhin National Medical Research Center of Oncology (N.N. Blokhin NMRCO) of the Ministry of 9
Health of the Russian Federation, 115478 Moscow, Russia; kisele@inbox.ru (M.K.) 10
3 Center for Biomedical Engineering, National University of Science and Technology “MISIS”, 119049 Mos- 11
cow, Russia; n.u.anisimova@gmail.com (N.A.); mvgorshenkov@gmail.com (M.G.) 12
4 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, 119991 Moscow, Russia; rybalchen- 13
kogv@lebedev.ru (G.R.) 14
* Correspondence: sdobatkin@imet.ac.ru 15

Abstract: In this study, high-pressure torsion (HPT) was used to deform austenitic 316LVM stainless 16
steel at 20 °C and 400 °C. The effects of HPT on the microstructure, mechanical properties, and 17
functional properties of the steel were investigated. By applying both HPT modes on 316LVM steel, 18
a nanocrystalline state with an average size of structural elements of ~46-50 nm was achieved. The 19
density of dislocations and twins present in the austenite phase varied depending on the specific 20
HPT conditions. Despite achieving a similar structural state after HPT, the deformation tempera- 21
tures used have different effects on the mechanical and functional properties of the steel. After HPT 22
at 20 °С the yield strength of 316L steel increased by more than 9 times up to 1890 MPa, and the 23
fatigue limit by more than 2 times up to 550 MPa, comparing to coarse-grained counter-parts. After 24
HPT at 20 °C, 316LVM steel exhibits better ductility, higher low-cycle fatigue resistance, greater 25
resistance to corrosion, and improved in vitro biocompatibility compared to processing at 400 °C. 26
The reasons for the deterioration of properties after HPT at 400 °C are discussed in the article. 27

Keywords: Stainless steel; high pressure torsion; ultrafine grained microstructure; twinning; me- 28
chanical properties; corrosion resistance; biocompatibility in vitro 29
30

Citation: To be added by editorial

staff during production. 1. Introduction 31

Academic Editor: Firstname Last- Austenitic stainless steels (SS) are widely used in various industrial applications and 32
name in the medical field due to their excellent combination of functional properties, including 33
high strength, ductility, corrosion resistance, biocompatibility, and good formability [1]. 34
Received: date
These materials offer a versatile solution for a range of applications, providing both me- 35
Revised: date
Accepted: date
chanical and chemical durability, while also meeting the requirements for medical devices 36

Published: date
and implants [2]. Due to its low carbon content and molybdenum alloying, austenitic 316L 37
stainless steel (SS) exhibits high resistance to various forms of corrosion. Additionally, its 38
high nickel content stabilizes the austenitic phase, resulting in non-magnetic behavior. 39
These properties, combined with its high processability, biocompatibility, and cost effec- 40
Copyright: © 2023 by the authors.
tiveness, make 316L SS an ideal material for internal fixation devices used in orthopedic 41
Submitted for possible open access
publication under the terms and
surgery, as well as for the production of stents used in cardiovascular procedures [3]. The 42

conditions of the Creative Commons

versatile nature of 316L SS allows for its wide application range in both industrial and 43
Attribution (CC BY) license medical fields. However, SS materials do have some limitations. For example, they have 44
(https://creativecommons.org/license a low yield strength, which significantly restricts their application range. Additionally, in 45
s/by/4.0/). the field of biomedical usage, the low fatigue strength of 316L SS is a disadvantage, as it 46

Metals 2023, 13, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/metals

Metals 2023, 13, x FOR PEER REVIEW 2 of 17

is one of the lowest among alloys used in orthopedics, and fatigue damage is a common 47
cause of implant failure [4]. 48
Since surgical grade 316L SS is a stable material that does not undergo phase trans- 49
formations during heat treatment and deformation, grain refinement is one of the few 50
strategies to increase the yield strength [5]. One effective method of grain refinement is 51
the severe plastic deformation (SPD) [6, 7]. Studies [8-12] have demonstrated improve- 52
ments in the mechanical and operational properties, including fatigue strength, by obtain- 53
ing an ultrafine-grained (UFG) structure in austenitic corrosion-resistant steels through 54
equal channel angular pressing (ECAP) method. Another method that allows the ultrafine 55
structure refinement without sample destruction is high-pressure torsion (HPT) [7]. This 56
method has already been tested on austenitic stainless steels, including 316L SS [13-22]. 57
All studies confirmed the achievement of a nanocrystalline state in 316L SS after HPT. 58
Some researchers [15] have also highlighted the occurrence of a martensitic γ → ε → α’ 59
transformation in the steel structure during HPT, which has resulted in a significant in- 60
crease in hardness up to 6000 MPa. Furthermore, it has been noted that hardness is more 61
sensitive to changes in grain size rather than changes in phase composition. On the con- 62
trary, in the study of the deformation mechanisms through HPT in 316L steel over a wide 63
temperature range from -196 °C up to 720 °C, Scheriau et al. [16] determined that only at 64
deformation temperatures below room temperature did the deformation-induced γ(fcc) 65
→ ε(hcp) transformation occur. At deformation temperatures above 450 °C, the dominant 66
mechanism was dislocation slip, while in the range below 450 °C to room temperature, 67
deformation twinning in the austenite phase was observed. Moreover, in many studies, 68
authors have noted the increase in hardness and strength both after post-deformation an- 69
nealing of 316L SS [17-20] and during deformation through HPT at elevated temperatures 70
[20]. 71
Wang et al. [17], using TEM analysis and specific electrical resistivity measurements, 72
investigated nanocrystalline plates of 316L stainless steel in the annealed austenitic state 73
obtained through HPT at 500°C for one hour. Thus, the study [17] concluded that the 74
strengthening of 316L SS occurred through a combination of nanostructuring through 75
HPT and precipitation of segregations during annealing. 76
In further research, Renk et al. [18, 19] confirmed the precipitation of segregations in 77
samples of 316L SS with a diameter of 35 mm and a height of 11 mm, which were 78
nanostructured using the quasi-constrained high-pressure torsion method under a pres- 79
sure of 3.60 GPa for 15 rotations. In the study [18], the authors demonstrated a 20% in- 80
crease in hardness of nanostructured 316L SS after annealing at 823 K for 30 minutes. After 81
comparing the results of mechanical testing and atom probe tomography (APT) data, it 82
was concluded that the increase in hardness of 316L SS was not caused by segregations of 83
dissolved elements. However, particles of dissolved elements or second phases stabilize 84
the nanocrystalline structure and, therefore, allow the annealing processes such as anni- 85
hilation and relaxation, which are necessary for strengthening phenomena during anneal- 86
ing. These particles play a role in maintaining the stability and enhancing the mechanical 87
properties of the nanocrystalline material. In a subsequent study [19], the same authors 88
attempted to improve the fatigue limit of 316L SS by subjecting it to annealing at 823 K for 89
90 minutes, since this treatment resulted in the greatest increase in strengthening. During 90
the experiment, the fatigue limit increased up to 1 GPa. The authors concluded that the 91
increase was due to defect relaxation, which shifted the onset of plasticity to higher stress 92
levels, combined with the stabilization of nanograin boundaries through segregations 93
In the study [20], 316 steel was nanostructured using the HPT method at room tem- 94
perature and at 400 °C. As a result, in the austenitic steel at different temperatures of HPT 95
an ultrafine-grained structure was obtained. It was characterized by varying grain size, 96
dislocation density, and twinning density and exhibited a similar level of enhanced 97
strength of approximately 1700 MPa. Furthermore, the use of atom probe tomography 98
(APT) revealed that HPT at elevated temperatures resulted in the formation of Mo-Cr-Si 99
segregations in 316 stainless steel. Taking into account the various contributions to the 100
Metals 2023, 13, x FOR PEER REVIEW 3 of 17

material strengthening, the authors demonstrated that segregations can lead to a signifi- 101
cant increase in strength. By impeding dislocation glide, segregations also enhance the 102
yield strength. Since the initiation of fatigue cracks is a result of dislocation glide, HPT at 103
400 °C is expected to result in a significant increase in fatigue limit. 104
The aim of this study was to investigate the effect of nanostructuring using HPT 105
method at two temperatures, room temperature and 400 °C, on the operational character- 106
istics of surgical-grade 316LVM stainless steel, including fatigue strength, corrosion re- 107
sistance, and in vitro biocompatibility. The promising results [20] raised hopes for an im- 108
provement not only the specific strength of surgical-grade steel after HPT at 400 °C but 109
also fatigue strength while maintaining or even improving corrosion resistance and in 110
vitro biocompatibility. 111

2. Materials and Methods 112

The study utilized an austenitic 316LVM stainless steel with low carbon content, 113
which is commonly used for implant manufacturing. The nominal composition of the 114
316LVM steel is presented in Table 1. The material was initially in the as-received state 115
and was subjected to austenitization at 1050°C for 1 hour, followed by quenching in water. 116
High-pressure torsion (HPT) deformation was conducted using a Bridgman-type setup 117
on samples with a diameter of 20 mm and a thickness of 1.5 mm, applying a pressure of 6 118
GPa. Deformation was performed using two isothermal deformation modes: at tempera- 119
tures of 20 °C and 400 °C. For all deformation modes, the samples were subjected to 10 120
rotations, which corresponds to a true strain of approximately 5.7 at the center of the sam- 121
ple radius. 122

Table 1. Chemical composition of the 316LVM stainless steel. 123

Amount (wt.%)
Elements
C Cr Ni Cu Si Mn Mo N S,P Fe
316LVM 0.012 17.3 14.11 0.07 0.24 1.77 2.75 0.07 0.001/0.024 balance
124
TEM analysis of the structure was conducted using a JEM-200CX microscope, while 125
X-ray diffraction (XRD) analysis was performed using a DRON 4.07 diffractometer. For 126
the foils, samples were taken from the middle of the sample radius. Micro-hardness meas- 127
urements were conducted using a 402 MVD Wolpert Wilson instrument with a 1N load. 128
Mechanical properties were determined using an "INSTRON 3382" machine on flat sam- 129
ples that were 1.0 mm thick, 1.0 mm wide, and had a working length of 2.0 mm (Figure 130
1). High-cycle fatigue tests were performed under repeated tension conditions on an IN- 131
STRON 8801 servo-hydraulic machine with a maximum load capacity of 100 kN, a testing 132
frequency of 40 Hz, and a stress ratio of R = 0.1 on flat samples 1.0 mm thick, 1.0 mm wide 133
and length of working part of 1.6 mm 134
Metals 2023, 13, x FOR PEER REVIEW 4 of 17

135
Figure 1. Schematic representation showing the locations of all investigation and test samples on 136
the HPT disk. 137

To assess the impact of HPT processing on the biocompatibility of 316LVM SS, the 138
level of hemolysis and cytotoxicity relative to non-transformed immunocompetent blood 139
cells was evaluated. For the evaluation of hemolytic activity, the 316LVM steel samples 140
were incubated in 2 ml of Hank's solution containing 14.8·106 human erythrocytes at 37 141
°C in an atmosphere with 5% carbon dioxide. The incubation duration was 2, 4, and 24 142
hours. The level of hemolysis was determined according to a previously described method 143
[23]. 144
To study cytotoxicity, the steel samples were incubated in 2 ml of complete growth 145
medium containing 540,000 mononuclear leukocytes isolated from the venous blood of a 146
healthy donor using a Ficoll gradient. The incubation was performed at 37 °C in an atmos- 147
phere containing 5% carbon dioxide for 24 hours. In the control group, cells were incu- 148
bated in complete growth medium under the same conditions. The influence of the steel 149
on tumor cell viability was assessed by calculating the ratio of cell activity on the surface 150
of the samples to the control in the MTT assay, as described previously [24]. 151
For cell colonization experiments, a human MMSC culture (from the cell culture col- 152
lection of the N.N. Blokhin National Medical Research Center of Oncology) consisting of 153
20,000 cells was applied to the surface of the alloy samples after forging and after HPT, 154
with a volume of 15 μL. The samples were incubated for 30 minutes at 37°C in an atmos- 155
phere with 5% carbon dioxide. After incubation, 1 mL of complete growth medium was 156
added to the samples containing cells, and they were then incubated for 21 days under 157
the same conditions. In the control group, cells were incubated on the bottom of the well 158
plate under the same conditions. The growth medium was changed every 2 days. 159
Cell colonization was studied using fluorescence microscopy with a LionHeart LX 160
digital microscope (Perkin Elmer, USA) after staining the cells with Calcein AM (Sigma, 161
USA) following the manufacturer's instructions. At the end of the experiment, the alloy 162
samples were removed from the well plate where incubation took place and treated with 163
1 mL of trypsin. In the control group, the growth medium was removed and the cells were 164
treated with trypsin. Cell viability was assessed using The Muse Count & Viability Kit 165
(Muse, Thermo Scientific, USA) and the Muse cell analyzer (Millipore, Germany). To 166
study the expression of adhesion molecules by the cells, they were stained with antibodies 167
against CD44 (Bioscience, USA) and CD11b (Bioscience, USA), and the number of 168
CD44(+), CD11b(+), and CD44(+)CD11b(+) cells was determined using a NovoCyte flow 169
cytometer (ACEA Bioscience, USA) based on the analysis of at least 10,000 cells in the 170
sample. 171
The results of the research were presented as the mean value and standard deviation 172
(mean ± SD). To perform comparative analysis with the control groups, a t-test was used. 173
Metals 2023, 13, x FOR PEER REVIEW 5 of 17

Differences were considered statistically significant at p < 0.05. The experiments and pro- 174
cedures with cells and animals were assessed and approved by the Local Ethics Commit- 175
tee of “N. N. Blokhin National Medical Research Center of Oncology” of the Health Min- 176
istry of Russia (#8-03, 01/03/2023). 177
178

3. Results 179

3.1. Microstructural characterization of the 316LVM stainless steel in the initial state and after 180
HPT 181
Figure 2 shows the initial structure of the 316LVM stainless steel in the forged (Figure 182
1a) and quenched (Figure 2b) states. 183

184
Figure 2. Optical micrographs of the 316LVM stainless steel after forging (a) and quenching (b). 185

After HPT at room temperature and at 400 °C, a nanocrystalline structure with pre- 186
dominantly high-angle grain boundaries is formed in 316LVM steel. The presence of a 187
grain structure was determined by the circular electron diffraction pattern with point re- 188
flections. HPT at room temperature resulted in a microstructure, with an average size of 189
structural elements about 46 ± 1.8 nm and deformation twins in the austenitic matrix. With 190
an increase in HPT temperature up to 400 °C, the character of the forming structure re- 191
mains the same, and the average size of structural elements increases slightly to 50 ± 1.6 192
nm. Moreover, the density of twins after HPT at 400 °C, according to TEM analysis, is four 193
times higher than one at 20 °C. Deformation twinning typically occurs when slip is hin- 194
dered for various reasons. 195
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196
Figure 3. TEM images of the 316LVM stainless steel after HPT at 20°C (a,b) and 400°C: (c,d) with 197
SAED pattern can be seen in the inset of (a) and (c). 198

3.2. X-ray analysis of the 316LVM stainless steel after HPT 199
X-ray structural quantitative phase analysis revealed the presence of a fully austenitic 200
structure in the steel, deformed under all processing regimes (Figure 4, Table 2). By ana- 201
lyzing the width of the X-ray lines of the austenite (111) and (222) reflections, the param- 202
eters of the fine crystalline structure of the 316LVM stainless steel after HPT were deter- 203
mined. This included the size of coherent scattering regions (CSR) and the root mean 204
square microstrain, which further allowed the calculation of dislocation density. The line 205
profiles were fitted using Gaussian functions. The X-ray structural analysis did not reveal 206
any significant differences in the sizes of CSR (30-40 nm) and showed a twofold decrease 207
in dislocation density after increasing the deformation temperature from room tempera- 208
ture to 400 °C. he values of microstrains decrease with an increase in the HPT temperature. 209
The highest value of microstrain was obtained after HPT at room temperature. The de- 210
crease in CSR values corresponds to a reduction in grain size observed through electron 211
microscopy analysis. However, there is only a slight difference between the average size 212
of structural elements determined by electron microscopy and the CSR size determined 213
by X-ray structural analysis. This discrepancy may be attributed to the fact that X-ray 214
structural analysis takes into account subgrain structures that are not always detectable 215
through electron microscopy [25]. 216
Metals 2023, 13, x FOR PEER REVIEW 7 of 17

217
Figure 4. XRD patterns of the 316LVM stainless steel after HPT. 218

Table 2. Results of the X-ray phase analysis of the 316LVM stainless steel after HPT. 219

Processing Space group Phase a, Å Content, wt.%

HPT, at 20 °С 225: Fm-3m γ 3.596 100.0 ± 0.0

HPT, at 400 °С 225: Fm-3m γ 3.599 100.0 ± 0.0
220

Table 3. Сrystallite size, microstrain and dislocation density of the 316LVM stainless steel after HPT 221
determined by X-ray line profile analysis. 222

Processing 111 ,° 222,° d, nm1 ε, %2 ρ, 1015 (m−2) 3

HPT, at 20 °С 0.82  0.08 2.56  0.25 31.4  1.52 0.33  0.10 143.2
HPT, at 400 °С 0.60  0.06 1.83  0.18 41.5  1.89 0.23  0.07 75.4
1 d - crystallite size, ε – microstrain, ρ - dislocation density.
2 3 223

3.3. Microhardness of the 316LVM stainless steel after HPT 224

The degree of deformation during HPT increases with the distance from the center 225
along the radius, resulting in smaller sizes of the structural elements towards the edge of 226
the sample and higher microhardness values. All investigated samples after HPT exhib- 227
ited lower microhardness values in the center of the sample (4.2-5.0 GPa) and an increase 228
in microhardness at the edge of the sample, reaching 5.0-5.5 GPa (Figure 5a). The samples 229
subjected to HPT at room temperature showed more uniform deformation with the small- 230
est difference in microhardness between the center and the edge of the sample. 231
Figure 5 illustrates the distribution of microhardness over the surface of an HPT sam- 232
ple after HPT from the side of the plunger (Figure 5b, d) and support (Figure 5c, e). The 233
microhardness values from the support side are higher and more uniformly distributed 234
across the surface. Microhardness measurements across the sample surface visually 235
demonstrated not only a significant difference in microhardness values between the cen- 236
ter and edges of the samples after HPT at 400 °C, but also higher microhardness values in 237
these samples at the established stage with the same microhardness (1/2 R - R from the 238
center of the sample). 239
Metals 2023, 13, x FOR PEER REVIEW 8 of 17

240
Figure 5. Microhardness of the 316LVM stainless steel produced by HPT: (a) along the diameter; (b 241
- d) distribution over surface of an HPT sample after HPT at 20 °C (b, c) and after HPT at 400 °C (d, 242
e), from the side of the plunger (b, d) and support (c, e). 243

Investigation of the thermal stability of the steel samples after severe plastic defor- 244
mation (SPD) revealed that the deformation strengthening for all modes decreases at tem- 245
peratures above six hundred and fifty degrees, due to grain growth (Figure 4). In the tem- 246
perature range of 400-650 °C, there is a slight increase in microhardness, with the growth 247
in microhardness starting slightly earlier and being more pronounced at a deformation 248
temperature of 400 °C. 249

250
Figure 6. Microhardness of the 316LVM stainless steel produced by HPT as a function of annealing 251
temperature. 252

3.4. Mechanical properties under static and cyclic loading 253

HPT significantly enhances the strength characteristics of 316LVM steel (Figure 7, 254
Table 4). The yield strength increases by more than 9 times, while the ultimate tensile 255
strength increases by 4 times. However, the ductility decreases after HPT at 20 °C to ε = 256
11%, and sharply drops to ε = 1% at a deformation temperature of 400 °C. At approxi- 257
mately similar average sizes of the structural elements, an increase in the HPT tempera- 258
ture results in a twofold decrease in the density of free dislocations and an increase in the 259
density of twins, which should not lead to a loss of ductility compared to the deformation 260
level at room temperature. 261
Metals 2023, 13, x FOR PEER REVIEW 9 of 17

262
Figure 7. Stress–strain curve of the 316LVM stainless steel after quenching and HPT. 263

264
Table 4. Mechanical properties of the 316LVM stainless steel after quenching and HPT. 265

Processing σUTS [MPa]1 σYS [MPa]2 ε [%]3

HPT, at 20 °С 2005 ± 24 1890 ± 6 11± 0.45
HPT, at 400 °С 1960± 18 1820 ± 12 1 ± 0.3
Quenching 500 ± 4 200 ± 6.5 47.6 ± 1.5
1 ultimate tensile strength; 2 yield strength; 3 total elongation 266
267
The fatigue limit of the samples after HPT at room temperature (550 MPa) exceeds 268
the fatigue limit of the as-quenched sample (250 MPa) by more than twofold (Figure 8). 269
Previously, the authors of this study observed a significant increase in fatigue strength 270
due to grain refinement and twinning in austenite during severe plastic deformation 271
(SPD) and subsequent cyclic deformation [12]. 272

273
Figure 8. The Woehler (S-N) curves for cyclic deformation of the 316LMV stainless steel after 274
quenching and HPT. 275
Metals 2023, 13, x FOR PEER REVIEW 10 of 17

The results of the study on the samples after HPT at 400 °C did not reveal a decrease 276
in high-cycle fatigue, but showed a slight decrease in stress under low-cycle loading com- 277
pared to the samples after HPT at 20 °C. 278
The fractography of the surface of the as-quenched sample after fatigue failure indi- 279
cates that in the fatigue crack initiation and propagation zone (Figure 9a), a predominantly 280
ductile nature of the surface relief is observed. In certain areas, the relief appears quasi- 281
ductile with signs of fatigue striations (Figure 9b). In static fracture, typical ductile dimple 282
fracture is observed (Figure 9). 283

284
Figure 9. Fracture surfaces of fatigue failure of the 316LVM stainless steel samples after quenching 285
(a,b,c), HPT at RT (d,e,f) and HPT at 400 °C (g,h,i). 286

After HPT, the relief in the fatigue crack initiation and propagation zone is less de- 287
veloped compared to the case of samples in the as-quenched state. A distinct zone of ac- 288
celerated crack growth before the static fracture can be clearly identified (Figure 9d,g). 289
The main crack propagation direction is shown along the striations. Signs of fatigue stria- 290
tions are observed (Figure 9d,h). The static fracture, like in the as-quenched samples, is 291
associated with a ductile dimple fracture mechanism (Figure 9f,i). Deeper dimples on the 292
fracture surface of the sample after HPT at room temperature indicate higher material 293
ductility compared to the samples after HPT at 400 °C. The presence of microcracks within 294
the dimples (Figure 9h) after HPT at 400 °C suggests the presence of grain boundary pre- 295
cipitates, which could have contributed to their initiation. 296
297
3.4. Corrosion resistance of the 316LVM stainless steel in the initial state and after HPT 298
Metals 2023, 13, x FOR PEER REVIEW 11 of 17

The results of potentiodynamic analysis on 316LMV stainless steel samples in the as- 299
quenched state and after HPT are presented in Figure 10. HPT at room temperature, based 300
on the polarization curves, shows minimal changes in the corrosion resistance (Figure 301
10a). 302

303
Figure 10. PDP curves in 0.1M NaCl (pH=6) electrolytes at scan rate of 1 mV/s (a) and E vs. corrosion 304
current density (i) (b). 305

The lower corrosion potential (Ecorr = -260 ± 51 mV) and higher corrosion current den- 306
sity (icorr = 0.406 ± 0.152 µ A/cm²) compared to the as- quenched state (Ecorr = -243 ± 47 mV, 307
icorr = 0.134 ± 0.088 µ A/cm²) and the state after HPT at 20 °C (Ecorr = -223 ± 48 mV, icorr = 308
0.189 ± 0.081 µ A/cm²) indicate a lower corrosion resistance of the samples after HPT at 400 309
°C (Figure 9b). 310

Table 5. Potentiodynamic polarization parameters of the 316LVM stainless steel. 311

Processing Ecorr 1, mV icorr 2, µA/cm2

Quenching, at 1050 °С (1 h) -243 ± 47 0.134 ± 0.088
HPT at 20 o C -223 ± 48 0.189 ± 0.081
HPT at 400 o C -260 ± 51 0.406 ± 0.152
1 corrosion potential; 2 corrosion current density 312
3.5. Biocompatibility in vitro of the 316LVM stainless steel in the initial state and after HPT 313
To assess the effect of deformation using the HPT method on the biocompatibility of 314
316LVM stainless steel, hemolysis levels were examined after incubation with the sam- 315
ples, as well as their cytotoxicity relative to non-transformed immune-competent blood 316
cells. According to recommendations [26,27], materials that induce hemolysis rates of no 317
more than 5% are considered biocompatible with low hemolytic activity. The hemolysis 318
levels were evaluated after 2, 4, and 24 hours of incubation relative to the spontaneous 319
hemolysis level in the control with intact cells. 320
The conducted studies showed that the hemolytic activity of all the examined sam- 321
ples did not exceed 2% throughout the entire observation period (Figure 11a). Further- 322
more, no significant difference in the impact on the hemolysis level was observed between 323
the alloy in its as-received state and after HPT. 324
The assessment of cytotoxicity of the steel (Figure 11b) through measuring the level 325
of cell activity using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bro- 326
mide) assay after 1-day incubation relative to the control with intact cells showed that the 327
presence of the examined samples, both in their as- quenched state and after HPT, did not 328
Metals 2023, 13, x FOR PEER REVIEW 12 of 17

lead to a significant change in cell activity level (p > 0.05). Thus, no signs of cytotoxic ef- 329
fects of 316LVM SS were found in this cell model. 330

331
Figure 11. The hemolysis (a)and cytotoxicity (b) of the 316LVM stainless steel samples after quench- 332
ing and HPT calculated as a percentage relative to control. 333

Since the examined samples of 316LVM SS in their as-quenched state and after HPT 334
did not induce hemolysis exceeding 2% and did not exhibit cytotoxicity, these samples 335
can be considered biocompatible. HPT did not lead to a significant deterioration in this 336
parameter. 337
To investigate the impact of processing on 316LVM SS on the colonization of mesen- 338
chymal stromal/stem cells (MSCs), the cells were incubated on the surface of the samples 339
in their as-quenched state, after HPT at room temperature, and after HPT at 400 °C for a 340
period of 21 days. The assessment of their viability on the surface of the samples compared 341
to the control (Figure 12) did not reveal any significant differences in the proportion of 342
viable cells compared to the control (p < 0.05) on the surface of the samples in their as- 343
quenched state and after HPT at 20 °C. However, on the surface of the samples after HPT 344
at 400 °C, there was a 1.2-fold decrease in the concentration of adhered viable cells com- 345
pared to the control (p=0.04). 346

347
Figure 12. Cell viability on the surface of the 316LVM stainless steel samples after quenching and 348
HPT relative to control. 349

The images allowing the evaluation of the number of live and dead cells colonizing 350
the 316LVM SS samples at the end of the incubation period indicate not only a decrease 351
in the ratio of live/dead cells on the surface of the 316LVM SS sample after HPT at 400 °C 352
Metals 2023, 13, x FOR PEER REVIEW 13 of 17

but also a suppression of cellular colonization overall (Figure 13). The concentration of 353
live cells on the surface of the 316LVM SS samples after HPT at 20 °C was approximately 354
comparable to or even slightly higher than that of the as-quenched state sample. At the 355
same time, it was observed that the majority of cells were not arranged individually but 356
rather in groups, forming colonies. This can be interpreted as evidence of an actively on- 357
going cell division process, accompanying the colonization of the surface of the samples. 358

359
Figure 13. Колонизация поверхности of the 316LVM stainless steel samples after quenching(a), 360
HPT at 20 °С (b) and HPT at 400 °С (c) by ММSC after 21 days of incubation. Cell staining Calcein 361
AM. 362

The next step was aimed to further investigate the mechanism underlying the ob- 363
served effect of processing of 316LVM SS on the cell colonization. In order to do this, the 364
change in the concentration of cells expressing the adhesion molecules CD11b and CD44 365
on the cell membranes, colonizing the surface of the steel, was studied in comparison to 366
the control (Figure 14). The transmembrane molecules of integrin CD11b are involved in 367
various aspects of cellular activity and influence cell attachment to the extracellular ma- 368
trix. The transmembrane glycoprotein molecules CD44, integrated into the cell membrane, 369
influence both cell adhesion to the extracellular matrix and intercellular interactions, pro- 370
moting cell migration. 371

372
Figure 14. The expression of adhesion molecules CD44 and CD11b on the membranes of MSCs col- 373
onizing the surface of the 316LVM SS samples in the initial state and after HPT was compared to 374
the control. 375

According to the obtained data, contact with the 316LVM SS samples stimulated the 376
expression of surface adhesion molecules CD44 and CD11b in MSCs compared to the con- 377
trol. Moreover, the impact of 316LVM SS after HPT at 20 °C was more pronounced com- 378
pared to the as-quenched sample and the sample after HPT at 400 °C. This effect was less 379
determined by the increasing expression of CD11b integrins on the cell surface and more 380
by the increasing expression of CD44. Thus, it can be concluded that the cell colonization 381
Metals 2023, 13, x FOR PEER REVIEW 14 of 17

of 316LVM SS samples is caused by the stimulation of synthesis and expression of trans- 382
membrane adhesion molecules by the cells. Moreover, the samples after HPT at 20 °C 383
stimulated colonization to the greatest extent through the involvement of this mechanism. 384

4. Discussion 385
In this study, surgical grade 316LVM stainless steel was investigated, which under- 386
goes vacuum arc remelting to increase its purity (lower phosphorus and sulfur content), 387
making it more corrosion-resistant and biocompatible compared to 316L. In terms of 388
chemical composition, 316LVM stainless steel differs slightly from 316L. It has an in- 389
creased lower limit for molybdenum, reduced silicon content (maximum allowable value 390
of 0.75% vs 1.0%), and a higher range for allowable chromium and nickel content accord- 391
ing to ISO 5832-1 standard. In addition, the microstructure should not contain molyb- 392
denum-enriched chi and sigma intermetallic compounds, which can lead to reduced cor- 393
rosion resistance and potential embrittlement. 394
In this study, two HPT modes, performed at room temperature and at 400 °C, re- 395
sulted in a nanocrystalline state in the 316LVM steel, with an average size of structural 396
elements of 46 ± 1.8 nm and 50 ± 1.6 nm, respectively. The structure differed only in the 397
density of twinning in the austenite (four times higher after HPT at 400 °C, as observed 398
using TEM) and the density of dislocations (twice higher after HPT at 20 °C, as measured 399
by X-ray). No precipitates were detected in the microstructure of the 316LVM steel using 400
TEM. The mentioned above distinguishing features of the microstructure do not explain 401
the similar levels of strength properties with a significant difference in ductility between 402
the two states after HPT at room temperature and at 400 °C (ε= 11 ± 0.45% and ε= 1 ± 0.3%, 403
respectively). Only the increased density of twins indicates that something hindered the 404
dislocation glide during the HPT process at 400 °C. 405
The reported in [17, 18, 19, 20] mechanism of the segregation of Mo-Cr-Si precipita- 406
tion, allow to suppose that increased content of Mo and Cr in the investigated 316LVM 407
steel could have led to more pronounced segregation. This had a negative impact not only 408
on the mechanical properties (Figure 7, Table 4) but also on the operational characteristics. 409
The results of fatigue tests showed a decrease in fatigue strength under low-cycle loading 410
(Figure 8). Moreover, fractographic analysis of the fracture surface of the sample after HPT 411
at 400 °C revealed the presence of microcracks (Figure 9h), indicating possible segregation 412
that could have been the cause of their initiation. The corrosion studies using potentiody- 413
namic polarization analysis revealed that HPT at 400 °C reduces the corrosion potential 414
of the surgical steel, and increases the corrosion current density of the samples (Figure 10, 415
Table 5). In vitro studies (Figure 11-14) of 316LVM stainless steel revealed lower biocom- 416
patibility indicators for samples after HPT at 400 °C. However, while the difference in the 417
hemolysis and cytotoxicity levels of the samples (Figure 11) was not significant (p > 0.05), 418
the colonization of MMSCs on the surface of the steel after HPT at 400 °C (Figure 12) 419
showed a decrease in the concentration of adhered living cells compared to the control 420
(p=0.04). Additionally, there was an overall suppression of cell colonization (Figure 13). 421
Thus, this study has established that the previously observed segregation of Mo-Cr- 422
Si in 316L stainless steel can significantly deteriorate the operational characteristics in sur- 423
gical-grade 316LVM steel with higher Mo and Cr content, negating the advantages of its 424
high-strength state. 425
Simultaneously, the 316LVM SS samples after HPT at 20 °C demonstrated a high 426
combination of properties, indicating not only high strength characteristics (σUTS = 2005 427
MPa and σYS = 1890 MPa) with satisfactory ductility for such a high-strength state (ε = 428
11%), but also high levels of fatigue strength, improved resistance to pitting corrosion, 429
and enhanced in vitro biocompatibility compared to the as-received state. It was deter- 430
mined that the cell colonization on the 316LVM SS samples was induced by the stimula- 431
tion of cell synthesis and expression of transmembrane adhesion molecules. This mecha- 432
nism was primarily stimulated by the 316LVM steel samples after HPT at 20 °C. The col- 433
onization of 316LVM SS after such processing could be influenced by both the surface 434
Metals 2023, 13, x FOR PEER REVIEW 15 of 17

morphology of the nanocrystalline material and its chemical composition. Both of these 435
factors can contribute to or inhibit changes in the local membrane potential of the cell in 436
the contact area, which stimulates protein synthesis and the formation of covalent bonds 437
with the cell membrane. In turn, the increase in the concentration of membrane-bound 438
molecules affects the formation of a tighter intercellular contact. This, in turn, enhances 439
the effectiveness of co-stimulation, leading to an increase in the functional potential of 440
MMSCs. It should also be noted that the inhibition of changes in the local membrane po- 441
tential of the cell may have been influenced by the segregation of Mo-Cr-Si, which resulted 442
in a deterioration of cell colonization in the steel after HPT at 400 °C. 443
The obtained data suggest that 316LVM SS after HPT at 20 °C can be considered as a 444
material for the development of submerged implants and metal structures for osteosyn- 445
thesis. These implants will effectively stimulate cell colonization on their surface and pro- 446
mote accelerated fixation in the area of bone defect repair, leading to enhanced cell differ- 447
entiation of pluripotent stromal cells. Additionally, they will contribute to the increase in 448
periosteal formation and local neo-osteogenesis. The utilization of HPT processing at 20 449
°C will not only significantly improve the operational characteristics but also reduce the 450
weight of medical devices. This is especially important in the development of implantable 451
prostheses for skeletal defect replacement, as it enables a significant increase in the specific 452
strength of the steels. Furthermore, the miniaturization of medical devices will result in a 453
reduced negative impact on the human body from the presence of foreign implants. 454
455

5. Conclusions 456
1. It was found that during HPT at room temperature and after HPT at 400 °C, the 457
316LVM steel forms a nanocrystalline structure with predominantly high-angle grain 458
boundaries and the presence of deformation twins in the austenitic matrix. 459
2. The achieved structure provides a level of microhardness that exceeds the micro- 460
hardness of the steel in the as-quenched state by more than 2.5 times. 461
3. HPT at room temperature and at 400 °C provides the same high level of strength 462
for 316LVM steel, with different levels of ductility of ε = 11% and ε = 1%, respectively. 463
4. HPT increases the fatigue limit of 316LVM steel by more than two times (up to 550 464
MPa) compared to the as-quenched state at a deformation temperature of 20 °C and re- 465
duces low-cycle fatigue at the deformation temperature of 400 °C. 466
467
5. The lower corrosion potential and higher corrosion current density of 316LVM 468
steel after HPT at 400 °C indicate a greater susceptibility to corrosion compared to after 469
HPT at 20 °C. 470
6. HPT at 20°C of 316LVM SS stimulates cell synthesis and expression of transmem- 471
brane adhesion molecules, leading to improved cell colonization on the surface of the 472
samples. This finding indicates the potential for utilizing nanocrystalline surgical steel. 473
474
Author Contributions: Conceptualization, O.R., M.K. and S.D.; methodology, N.A., N.M., G.R. and 475
M.K.; software, G.R., A.T., D.P. and M.G.; validation, O.R., N.A. and N.M.; formal analysis, O.R. and 476
N.M.; investigation, O.R., N.A., N.M., G.R., A.T., E.L., D.P. and M.G..; resources, A.T.; data curation, 477
A.T., E.L. and S.D.; writing—original draft preparation, O.R. and N.A.; writing—review and editing, 478
G.R., S.D., E.L., M.K. and N.M.; visualization, O.R., G.R., M.G., N.A. and E.L.; supervision, S.D. and 479
M.K.; project administration, O.R.; funding acquisition, S.D. All authors have read and agreed to 480
the published version of the manuscript. 481

Funding: This research was carried out with a support of the Russian Federation state assignment 482
of A. A. Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Science 483
(IMET RAS), Russia (Theme No. 075-01176-23-00) 484

Data Availability Statement: All the data required to reproduce these experiments are present in 485
the article. 486
Metals 2023, 13, x FOR PEER REVIEW 16 of 17

Acknowledgments: The study of fractography was carried out using research equipment of the 487
Shared Facility Center of the P.N. Lebedev Physical Institute of RAS "Center for the Study of High- 488
Temperature Superconductors and Other Strongly Correlated Electronic Systems". 489

Conflicts of Interest: The authors declare no conflict of interest. 490

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