Mechanical and in vitro biological properties of uniform and graded Cobalt‐chrome lattice structures in orthopedic implants

Abstract Human bones are biological examples of functionally graded lattice capable to withstand large in vivo loading and allowing optimal stress distribution. Disruption of bone integrity may require biocompatible implants capable to restore the original bone structure and properties. This study aimed at comparing mechanical properties and biological behavior in vitro of uniform (POR‐FIX) and graded (POR‐VAR) Cobalt‐chrome alloy lattice structures manufactured via Selective Laser Melting. In compression, the POR‐VAR equivalent maximum stress was about 2.5 times lower than that of the POR‐FIX. According to the DIC analysis, the graded lattice structures showed a stratified deformation associated to unit cells variation. At each timepoint, osteoblast cells were observed to colonize the surface and the first layer of both scaffolds. Cell activity was always significantly higher in the POR‐VAR (p < 0.0005). In terms of gene expression, the OPG/RANKL ratio increased significantly over time (p < 0.0005) whereas IL1β and COX2 significantly decreased (7 day vs 1 day; p < 0.0005) in both scaffolds. Both uniform‐ and graded‐porosity scaffolds provided a suitable environment for osteoblasts colonization and proliferation, but graded structures seem to represent a better solution to improve stress distribution between implant and bone of orthopedic implants.

these must sustain whilst preserving the physiological range of motion and the multiplanar mobility of the intact joint. In the lower limb, these implants must withstand large-magnitude dynamic loadings up to four times the body weight according to the motor task, 11 and must be wear-resistant in a biological environment. In case of joint replacement, the need for strong primary fixation of the implant with maximum preservation of the original bone stock, and for minimization of the stress shielding due to the different mechanical properties with respect to those of the bone, 12,13 has pushed the research for lattice graded materials with appropriate mechanical and osteointegration properties. [14][15][16][17][18] For orthopedic implant applications, uniform or graded porosity scaffolds can be obtained from the repetition of unit cells with different geometrical shapes and density as to mimic the radially graded porosity of the human long bones. 19 Due to its good mechanical properties and elasticity, titanium alloys have long been used for most orthopedic implants and fixation devices, with the exclusion of the load-bearing implants used for joint replacement for which Cobaltchrome alloy (CoCr) alloys are generally the materials of choice. The high modulus of elasticity of CoCr, around twice as large as that of titanium alloys, is a key property to provide implants with sufficient strength to bear physiological loadings but may result disadvantageous in terms of stress shielding. Therefore, design optimization of the implant-to-bone interface of CoCr endoprostheses is particularly critical. In addition to providing implants with the proper mechanical behavior, a porous interface allows for primary and long-term fixation to the hosting bone thus a good osteointegration should be guaranteed. Pores size, overall porosity and interconnectivity are critical properties affecting cells migration within the implant, promoting the growth and avoiding overcrowding, allowing the passage of nutrients and of oxygen supply, and removing metabolic waste. 10 While the optimal pore size of structures interacting with some biological tissues has been identified, the optimal porosity of the implant-to-bone interface of orthopedic implants is still controversial. [20][21][22] Nevertheless, it is now generally accepted that lattices with pore diameters between 300-1,000 μm provide bone cells with suitable environment for viability and proliferation, regardless of the unit cells type. 23 It has been observed that structures with pore size larger than 300 μm are advantageous in terms of cell proliferation and deep colonization, and these beneficial effects override the initial better cell attachment induced by smaller pores. 24 Despite the extensive literature on this topic, covering the optimal porosity 25,26 and the fatigue behavior of lattice structures also via topological modelling, 27,28 it is still unclear whether biomimetic graded scaffolds are advantageous with respect to uniform density scaffolds in terms of osteointegration and minimization of the stress shielding between implant and bone. Functionally graded lattice structures obtained by the repetition of unit cells of varying sizes and shapes according to the local functional request of the implant 29 should be exploited to improve osseointegration and to limit stress shielding failures. [30][31][32] Low density structures have already been shown to be apt for bone cells proliferation and, in terms of mechanical interaction, may help orthopedic implant and prosthesis components to better conform with the overall bone stiffness. A gradual increase of volumetric density, from the inner region of the implant to the external surface of the endoprosthesis, is a feasible design solution to adjust the mechanical properties promoting correct load and stress distribution between implant and bone.
While the effect of unit type and porosity on the mechanical properties and interaction with biological tissues in vitro and in vivo has been largely investigated for Titanium alloys scaffolds, 25,33-36 the current knowledge on mechanical and biological properties of functionally graded CoCr lattices is still limited. 19,37 This study aimed at providing novel information on the mechanical and biological behavior of CoCr lattice that may be used as material for orthopedic implants.
Moreover, we aimed at identifying possible differences between uniform-and variable-porosity scaffolds presenting the same material, unit cell and average porosity. The latter may be used to gradually decrease the stiffness of the implant interface closer to the bone, thus helping to decrease the stress shielding of endoprostheses.

| Design and manufacturing of the samples
The spherical hollow cubic unit type used to design the lattice scaffolds was chosen following a careful mechanical and biological analysis performed in a previous study. 38 This unit cell (Figure 1a Group, Roma, Italy) and pre-wetted with osteoblast growth medium for 1 day at 37 C. All tests were performed considering lattice samples in as-built conditions, without post-processing treatments, either mechanical or thermal. To assess biocompatibility, each porous sample was placed in 48-well plates to avoid cells' dispersion (as previously described), 38 statically seeded with 5 Â 10 4 cells suspended in 1 ml of medium, moved to a new 24-well plate after 1 day and maintained in culture until 14 days.

| Cell culture conditions
NHOst were also seeded directly in tissue-culture polystyrene wells as bidimensional standardized control (CTR). Medium was refreshed twice a week.

| Cell viability and proliferation
Cell viability was observed at 1 day, 7 day, and 14 day by Alamar blue assay (Serotec, Oxford, UK) as previously reported. 38 Samples immersed in culture medium, but without cells, were used as control for the background fluorescence.
In order to evaluate the proliferation, cells were washed with phosphatase buffer solution (PBS), detached by repeated pipetting with trypsin/EDTA (Sigma-Aldrich, UK), harvested in complete medium to stop the trypsin action, and counted in Neubauer chamber using the erythrosin vital dye, which stains the dead cells.

| Gene expression
Gene expression was observed at 1 day, 7 day and 14 day of culture.  temperature was 55 C for all the primer sets except for TNFSF11 and GAPDH (60 C and 56 C, respectively). The mean threshold cycle was determined for each sample and used for the calculation of relative expression using the Livak method (2 -ΔΔCt ), with GAPDH as reference gene and CTR samples at 24 hr as calibrators at each experimental time. 40 Statistical analysis was performed using R v.3.6.1 software 41 and R packages "lme4" v. 1.1-21, 42 "lmerTest" v.3.1 43 "emmeans" v.1.4.1, 44 and "ggplot2" v.3.1.1. 45 Normal distribution (Shapiro-Wilk normality test) and homogeneity of variance (Levene test) were verified before doing data analysis. Data are presented as boxplots or Mean ± SD at a significant level of p < 0.05. Linear mixed models (LMM) were used to evaluate if there were significant interactions or effects of "material" factor (between-subjects) and "experimental time" factor (within-subjects, repeated measures)-on cell vitality and proliferation, and gene expression.
Pairwise comparisons of estimated marginal means (also known as least-squares means) were carried out as post-hoc tests to identify significant differences among Groups in term of effect size d msw : 46  Table 1).
In order to compare the stress behaviour between the two scaf-    In both scaffolds, the cells were more prone to collapse in the sections parallel to the loading surface, where the stress was maximum.
In particular, the cell count related to the lattice samples showed a substantial stability over time, but always higher values on POR-VAR than POR-FIX, although not statistically significant (Figure 6b).
The increasing values over time observed in CTR group, significantly higher than those of POR-FIX and POR-VAR, are probably due to the wider available culture surface of the bottom well, considered the gold standard for cell culture and representing the internal control of the system.
Conversely to what observed for cell proliferation, cell activity on POR-VAR scaffolds at each timepoint was significantly higher than cell activity on POR-FIX. Furthermore, a regular trend of increase was observed for cells on POR-VAR between 1 day and 14 day, and only a partial increase, between 7 day and 14 day, for POR-FIX. (Figure 6a).

| Cell morphology and scaffold colonization
Cell spreading and scaffold colonization were appreciated at all experimental timepoints by FITC-conjugate phalloidin, useful to evidence the cell cytoskeleton. The present study also investigated how SLM produced CoCr scaffolds could elicit inflammatory reaction in osteoblasts and could affect the complex balance involving bone formation and resorption.
Regarding the fine crosstalk between osteoblasts and osteoclasts, it is widely known the crucial role of the RANKL/RANK/OPG system: nuclear factor-kappa B (NF-κB) ligand (RANKL) has a fundamental role because able to bind RANK receptor on preosteoclast membrane, so triggering osteoclast maturation, or osteoprotegerin (OPG): a decoy receptor that limits the biologic activity of RANKL, competing with it. 53 In the present study, the ratio of OPG/RANKL expression significantly increased in the control over time, but also in both scaffolds.
Furthermore, the larger OPG/RANKL ratio observed in the CoCr lattice samples with respect to control suggest that these structures are effective in promoting osteosynthesis. Aseptic mobilization, one of the main issues contributing to failure of endoprostheses, depends also on the fine biological system regulating osteosynthesis and osteolysis, 54,55 which in turn could be affected by a possible inflammatory response induced by free nanoparticles or material debris. 56 SLM technique, in fact, produces irregular surfaces due to resolution allowed by the laser spot diameter with respect to the powder size.
Although the main cellular inflammatory response can be generated by the presence of few μm sized debris, 57 the possible presence of unmelted CoCr powder could warrant further evaluation. Therefore, the expression of IL1β and COX2 could indicate an osteoblastmediated inflammatory response, triggered by the unmelted powder particles with micrometric and sub-micrometric diameter (less than 300 nm) present in as-built SLM components. These free particles could stimulate osteolysis 55 and should be further investigated with respect to the manufacturing technique. Indeed, 1 day after scaffolds seeding, osteoblasts showed a clear activation of IL1β and COX2 expression, which was not observed in the control.
At 7 day and 14 day, however, no significant difference in the expression of these genes was observed between control and porous samples, thus suggesting the capability of osteoblasts to acclimatise to the CoCr environment and restore a normal noninflammatory response.

| CONCLUSIONS
Following the compression tests, the CoCr graded lattice structure presented an equivalent maximum stress about 2.5 times lower than that in the uniform structure and appeared more deformable, with a stratified strain behaviour associated to its porosity and to the unit cell geometry. The stiffness of the entire structure or of specific regions can be optimized according to the application.
Both uniform and graded structures provide the osteoblasts with an environment suitable for adhesion and proliferation, capable to support a favorable OPG/RANKL ratio and a self-limiting gene expression of the analyzed inflammatory mediators IL1β and COX2.
Both lattice structures presented good biocompatibility properties, but graded structures seem to offer a better solution to improve the stress distribution between CoCr orthopedic implants and bone.

ACKNOWLEDGMENT
The authors are grateful to Ministry of Health (funds 5 Â 1000, year 2019).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.