Native Osseous CaP Biomineral Coating on a Biomimetic Multi-Spiked Connecting Scaffold Prototype for Cementless Resurfacing Arthroplasty Achieved by Combined Electrochemical Deposition

The multi-spiked connecting scaffold (MSC-Scaffold) prototype with spikes mimicking the interdigitations of articular subchondral bone is an essential innovation in surgically initiated fixation of resurfacing arthroplasty (RA) endoprosthesis components. This paper aimed to present a determination of the suitable range of conditions for the calcium phosphate (CaP) potentiostatic electrochemical deposition (ECDV=const) on the MSC-Scaffold prototype spikes to achieve a biomineral coating with a native Ca/P ratio. The CaP ECDV=const process on the MSC-Scaffold Ti4Al6V pre-prototypes was investigated for potential VECDfrom −9 to −3 V, and followed by 48 h immersion in a simulated body fluid. An acid–alkaline pretreatment (AAT) was applied for a portion of the pre-prototypes. Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) studies of deposited coatings together with coatings weight measurements were performed. Themost suitable VECD range, from −5.25 to −4.75 V, was determined as the native biomineral Ca/P ratio of coatings was achieved. AAT increases the weight of deposited coatings (44% for VECD = −5.25 V, 9% for VECD = −5.00 V and 15% for VECD = −4.75 V) and the coverage degree of the lateral spike surfaces (40% for VECD = −5.25 V, 14% for VECD = −5.00 V and 100% for VECD = −4.75 V). XRD confirmed that the multiphasic CaP coating containing crystalline octacalcium phosphate is produced on the lateral surface of the spikes of the MSC-Scaffold. ECDV=const preceded by AAT prevents micro-cracks on the bone-contacting surfaces of the MSC-Scaffold prototype, increases its spikes’ lateral surface coverage, and results in the best modification effect at VECD = −5.00 V. To conclude, the biomimetic MSC-Scaffold prototype with desired biomineral coating of native Ca/P ratio was obtained for cementless RA endoprostheses.


Introduction
Resurfacing joint endoprostheses, e.g., total resurfacing hip arthroplasty (THRA) endoprostheses, are a bone-tissue-preserving option offered for relatively young and active patients with advanced osteoarthritis (OA). In contrast to traditional long-stem total hip replacement (THR) endoprostheses transform the amorphous CaP coating into bone-like biomineral coating) [54]. It was observed that the deposition of CaPs on surfaces of the MSC-Scaffold pre-prototypes can be controlled by adjusting the current density. These modifications were successfully performed in the galvanostatic ECD j=const process, but continued research using the potentiostatic process (ECD V=const ) showed significantly higher repeatability than the galvanostatic ECD process [55]. Similarly, the poorly investigated CaP deposition on complex-shaped substrates (e.g., scaffolds) and the unsatisfactory attempts during our preliminary research in applying ECD process parameters recommended for flat substrates to the MSC-Scaffold pre-prototypes, strongly justify the need to take an experimental approach for finding the suitable range of conditions for the CaP ECD V=const process on the MSC-Scaffold pre-prototypes.
The particular aim of this paper was to present the determination of the suitable range of conditions for potentiostatic electrochemical deposition of calcium phosphates (CaPs) on the MSC-Scaffold prototypes to achieve a native biomineral Ca/P ratio in the coating, which is of great importance for good biocompatibility and biological performance of the implant in vivo. Good biological performance of this implant was proved in our recent investigation in swines on a partial knee arthroplasty (RKA) endoprosthesis working prototype with the CaP coated in potentiostatic ECD process MSC-Scaffold [56].
The main aim of our work is to elaborate on the suitable MSC-Scaffold prototypes for a new generation of entirely cementless RA endoprostheses.

MSC-Scaffold Pre-Prototypes
Surface modification of the MSC-Scaffold prototype for non-cemented resurfacing joint endoprostheses was carried out on the MSC-Scaffold pre-prototypes designed as fragments of the central part of the femoral component of the TRHA endoprosthesis. The multilateral spikes of the MSC-Scaffold pre-prototypes were arranged in concentric parallel rings around the central spike with axes parallel to each other, whereas the central spike was coincident with the femoral head axis of symmetry. The length of the square side in the spike pyramid's base was 0.5 mm in the MSC-Scaffold CAD model. The prototype THRA endoprosthesis with the MSC-Scaffold manufactured using selective laser melting (SLM) of Ti4Al6V powder is presented in Figure 1A. In Figure 1B, CAD models of the MSC-Scaffold pre-prototypes designed for this research are presented. The two design variants vary by the distance between spike bases, 200 µm (P Sc200 ) and 350 µm (P Sc350 ), both circumferentially and radially, which corresponds to the thickness of bone trabeculae of cancellous bone. In Figure 1C, the MSC-Scaffold pre-prototypes manufactured using SLM are shown. The manufacturing was subcontracted to the Centre of New Materials and Technologies at the West Pomeranian University of Technology in Szczecin, Poland. The process parameters applied during the SLM manufacturing were: laser power 100 W, layer thickness 30 µm, laser spot size 0.2 mm, scan speed 0.4 m/s and laser energy density 70 J/mm 3 .
Materials 2019, 12, x FOR PEER REVIEW 3 of 15 (to transform the amorphous CaP coating into bone-like biomineral coating) [54]. It was observed that the deposition of CaPs on surfaces of the MSC-Scaffold pre-prototypes can be controlled by adjusting the current density. These modifications were successfully performed in the galvanostatic ECDj=const process, but continued research using the potentiostatic process (ECDV=const) showed significantly higher repeatability than the galvanostatic ECD process [55]. Similarly, the poorly investigated CaP deposition on complex-shaped substrates (e.g., scaffolds) and the unsatisfactory attempts during our preliminary research in applying ECD process parameters recommended for flat substrates to the MSC-Scaffold pre-prototypes, strongly justify the need to take an experimental approach for finding the suitable range of conditions for the CaP ECDV=const process on the MSC-Scaffold pre-prototypes.
The particular aim of this paper was to present the determination of the suitable range of conditions for potentiostatic electrochemical deposition of calcium phosphates (CaPs) on the MSC-Scaffold prototypes to achieve a native biomineral Ca/P ratio in the coating, which is of great importance for good biocompatibility and biological performance of the implant in vivo. Good biological performance of this implant was proved in our recent investigation in swines on a partial knee arthroplasty (RKA) endoprosthesis working prototype with the CaP coated in potentiostatic ECD process MSC-Scaffold [56].
The main aim of our work is to elaborate on the suitable MSC-Scaffold prototypes for a new generation of entirely cementless RA endoprostheses.

MSC-Scaffold Pre-Prototypes
Surface modification of the MSC-Scaffold prototype for non-cemented resurfacing joint endoprostheses was carried out on the MSC-Scaffold pre-prototypes designed as fragments of the central part of the femoral component of the TRHA endoprosthesis. The multilateral spikes of the MSC-Scaffold pre-prototypes were arranged in concentric parallel rings around the central spike with axes parallel to each other, whereas the central spike was coincident with the femoral head axis of symmetry. The length of the square side in the spike pyramid's base was 0.5 mm in the MSC-Scaffold CAD model. The prototype THRA endoprosthesis with the MSC-Scaffold manufactured using selective laser melting (SLM) of Ti4Al6V powder is presented in Figure 1a. In Figure 1b, CAD models of the MSC-Scaffold pre-prototypes designed for this research are presented. The two design variants vary by the distance between spike bases, 200 μm (PSc200) and 350 μm (PSc350), both circumferentially and radially, which corresponds to the thickness of bone trabeculae of cancellous bone. In Figure 1c, the MSC-Scaffold pre-prototypes manufactured using SLM are shown. The manufacturing was subcontracted to the Centre of New Materials and Technologies at the West Pomeranian University of Technology in Szczecin, Poland. The process parameters applied during the SLM manufacturing were: laser power 100 W, layer thickness 30 μm, laser spot size 0.2 mm, scan speed 0.4 m/s and laser energy density 70 J/mm 3 .  endoprosthesis with the multi-spiked connecting scaffold (MSC-Scaffold) manufactured using selective laser melting (SLM) of Ti4Al6V powder; (B) CAD models of the MSC-Scaffold pre-prototypes for RHA endoprostheses designed in two geometrical configuration variants, which vary by the distance between the spike bases, 200 µm (P Sc200 ) and 350 µm (P Sc350 ), both circumferentially and radially, and (C) the MSC-Scaffold pre-prototypes manufactured on the basis of these CAD models using SLM.

Preparation of the MSC-Scaffold Pre-Prototypes' Surfaces
After SLM manufacturing, to remove the adhered powder aggregates from the spike surfaces, a manual blasting treatment was carried out using an experimentally customized abrasive mixture composed of equal proportions of white aloxiteF220 (~53-75 µm), white aloxite F320 (~29.2 µm ± 1.5%), and blasting micro glass beads (~30 µm ± 10%) [57]. Cleaning in an ultrasonic bath (Sonic 3, Polsonic, Poland) was applied using the following agents distilled water, ethanol, acetone and, again, distilled water three more times; each stage of cleaning was carried out for 15 min. After that, the MSC-Scaffold pre-prototypes were dried at room temperature and the initial weight was measured using a precise analytical balance (AS 110/X, Radwag, Poland).

Determination of the Most Suitable Range of Conditions for the ECD V=const Process
To determine the most suitable range of potential (V ECD ) for the ECD V=const process, a total of 56 MSC-Scaffold pre-prototypes (28 of each variant) were subjected to surface modification. To search for the appropriate conditions of the ECD V=const process, V ECD values from −9 to −3V were investigated using a potentiostat-galvanostat apparatus (PGSTAT 302N, Metrohm Autolab, Ultrecht, The Netherlands). The CaP ions were deposited from a solution composed of 0.042 M calcium nitrate, Ca(NO 3 ) 2 , and 0.025 M ammonium dihydrogen phosphate, NH 4 H 2 PO 4 , with pH=6. The ECD process was performed in a two-electrode system. The process was carried out for one hour at room temperature. A gold plate anode was used as the counter electrode. After the ECD V=const process, the MSC-Scaffold pre-prototypes, playing the role of working electrode, were rinsed with distilled water and, to convert the deposited amorphous CaP coating into the bone-like biomineral coating, they were immersed for 48 h in an SBF solution composed of 6.8 g/L NaCl, 0.4 g/L KCl, 0.2 g/L CaCl 2 , 0.2048 g/L MgSO 4 ·7H 2 O, 0.1438 g/L NaH 2 PO 4 ·H 2 O and 1.0 g/L NaHCO 3 at 37 • C. After the incubation in SBF, the MSC-Scaffold pre-prototypes were dried at room temperature and their final weight was measured. The weight increase due to surface coatings deposited on the spikes of the MSC-Scaffold pre-prototypes was calculated as the difference between the initial and final weights of the modified MSC-Scaffold pre-prototype. An analysis of chemical composition of the coating deposited on the lateral spike surfaces of the MSC-Scaffold pre-prototypes was performed using a scanning electron microscope (Hitachi TM-3030, Hitachi High-Tech Technologies Europe GmbH, Krefeld, Germany) equipped with the energy dispersive X-Ray (EDS) system (Oxford Instruments, Abingdon, UK). The results can be used for calculating the Ca/P ratios.

The Influence of the AAT Pretreatment
The same ECD V=const surface modification process was performed to examine the influence of the AAT pretreatment on the final surface modification. In this step, the V ECD values that provided the highest weight increase simultaneous with Ca and P contents with Ca/P ratios corresponding to the Ca/P ratios of native osseous CaP there were applied to new pre-prototypes. A total number of 36 MSC-Scaffold pre-prototypes were modified, 12 for each V ECD value identified as favorable; half of the pre-prototypes underwent AAT pretreatment. The AAT process was conducted in 40% H 2 SO 4 for 40 min at 60 • C and subsequently in 1 mol/L NaOH for 40 min at 80 • C.
EDS surface mapping of three randomly selected subareas of the lateral spike surfaces of each MSC-Scaffold pre-prototype was performed using a specialized software analyser in the EDS system used. Based on mapping analysis, the regions with CaP deposited on the spikes' lateral surface were indicated and the coverage degree of the lateral spike surfaces was determined. In each of the analysed subareas, 10 pointwise measurements of the chemical composition were made, and the Ca/P ratios were calculated. The analyses of the coverage degree of the lateral surface of spikes and the deposited coating uniformity were made using the professional software tool ImageJ (National Institutes of Health, Bethesda, Maryland, USA). Structure and phase composition of the deposited coating was identified by XRD on a PANalytical EMPYREAN X-ray diffractometer (Malvern, UK) at a scanning speed of 0.02 • /s with Cu Kα radiation (λ = 0.15405 nm, 40 mA, 40 kV) at a 2θ range of 30-70 • . Since, there was no technical possibility to analyze the surface of MSC-Scaffold pre-prototypes directly, so to obtain the XRD roentgenograms we had to use the deposits detached mechanically from the spikes as a powder sample. Figure 2 shows a diagram of the mass increase due to the surface coating for the P Sc200 and the P Sc350 MSC-Scaffold pre-prototypes as a function of the applied V ECD values during the ECD V=const process of CaP deposition. coating was identified by XRD on a PANalytical EMPYREAN X-ray diffractometer (Malvern, UK) at a scanning speed of 0.02°/s with Cu Kα radiation (λ = 0.15405 nm, 40 mA, 40 kV) at a 2θ range of 30-70°.Since, there was no technical possibility to analyze the surface of MSC-Scaffold pre-prototypes directly, so to obtain the XRD roentgenograms we had to use the deposits detached mechanically from the spikes as a powder sample. Figure 2 shows a diagram of the mass increase due to the surface coating for the PSc200 and the PSc350MSC-Scaffold pre-prototypes as a function of the applied VECD values during the ECDV=const process of CaP deposition. For the PSc200MSC-Scaffold pre-prototypes modified using VECD values ranging from −9 to −5.25 V, the surface weight increase was about 3 mg for the initial stages (from 2.75 mg for VECD = −9 V to 3.65 mg for VECD = −7 V). Increasing the VECD value past −5.25 V resulted in reducing the deposited coating weight increase to 2 mg (for VECD = −4.75 V), while for VECD = −4.5 V and VECD = −3 V there was no noted weight increase. EDS analysis of chemical composition confirmed the absence of Ca and P on the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by applying VECD values of −4.5 V and −3 V. SEM analysis revealed that, for the PSc200 MSC-Scaffold pre-prototypes for which a weight increase was observed, CaPs were deposited only on the upper regions of spikes. In this case, a significant amount of CaP deposit was found in the inter-spike space of the MSC-Scaffold pre-prototypes. This phenomenon was judged to be disadvantageous. Example SEM photographs showing this effect are presented in Figure 3. None of the Ca/P ratios determined for the lateral spike surfaces of the PSc200 MSC-Scaffold pre-prototypes corresponds to the Ca/P ratio characteristic for CaPs. EDS analysis shows that the Ca/P ratios reached values below 1.00 and 3.73, so in this case, there was no CaP coating on the spike surfaces, but only Ca and P ions randomly deposited onto the surface of the MSC-Scaffold pre-prototypes' spikes. In the first case, almost the entire surface was deposited with Ca whereas in the second case, nearly all deposits were P. At −4.5 V no mass increase was observed. For the P Sc200 MSC-Scaffold pre-prototypes modified using V ECD values ranging from −9 to −5.25 V, the surface weight increase was about 3 mg for the initial stages (from 2.75 mg for V ECD = −9 V to 3.65 mg for V ECD = −7 V). Increasing the V ECD value past −5.25 V resulted in reducing the deposited coating weight increase to 2 mg (for V ECD = −4.75 V), while for V ECD = −4.5 V and V ECD = −3 V there was no noted weight increase. EDS analysis of chemical composition confirmed the absence of Ca and P on the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by applying V ECD values of −4.5 V and −3 V. SEM analysis revealed that, for the P Sc200 MSC-Scaffold pre-prototypes for which a weight increase was observed, CaPs were deposited only on the upper regions of spikes. In this case, a significant amount of CaP deposit was found in the inter-spike space of the MSC-Scaffold pre-prototypes. This phenomenon was judged to be disadvantageous. Example SEM photographs showing this effect are presented in Figure 3. None of the Ca/P ratios determined for the lateral spike surfaces of the P Sc200 MSC-Scaffold pre-prototypes corresponds to the Ca/P ratio characteristic for CaPs. EDS analysis shows that the Ca/P ratios reached values below 1.00 and 3.73, so in this case, there was no CaP coating on the spike surfaces, but only Ca and P ions randomly deposited onto the surface of the MSC-Scaffold pre-prototypes' spikes. In the first case, almost the entire surface was deposited with Ca whereas in the second case, nearly all deposits were P. At −4.5 V no mass increase was observed. the pre-prototype material, like Ti, V, and Al, are located on the lateral surface of the spikes (as is O, which is not shown) while Ca and P are distributed only as deposits in the inter-spike space. This phenomenon could be explained by considering the distance between the spikes. It is most likely caused by insufficient room between the spikes. The unwanted result of the CaP ECDV=const surface modification of the PSc200 MSC-Scaffold pre-prototypes led to the decision to abandon further research using this geometrical variant of MSC-Scaffold pre-prototype.  For the PSc350 MSC-Scaffold pre-prototypes modified using VECD values ranging between −9 to −5.50 V, the weight increase of the surface coating was low (less than 1 mg) and the Ca/P ratios determined in the deposited surface coatings did not correspond to the Ca/P values characteristic for CaPs. A significant increase in weight (about 5 mg) was found when applying VECDvalues ranging between −5.25 to −4.75V. For the ECD process carried out using VECD values above −4.50 V, a slight weight increase was observed (approximately 0.50-0.75 mg). Unfortunately, the Ca/P ratio in the deposited surface coating did not correspond to the characteristic Ca/P values for native osseous An example of the EDS chemical mapping of two magnified areas is presented in Figure 4 in which colors represent individual elements. As is clearly seen, the elemental species coming from the pre-prototype material, like Ti, V, and Al, are located on the lateral surface of the spikes (as is O, which is not shown) while Ca and P are distributed only as deposits in the inter-spike space. This phenomenon could be explained by considering the distance between the spikes. It is most likely caused by insufficient room between the spikes. The unwanted result of the CaP ECD V=const surface modification of the P Sc200 MSC-Scaffold pre-prototypes led to the decision to abandon further research using this geometrical variant of MSC-Scaffold pre-prototype. An example of the EDS chemical mapping of two magnified areas is presented in Figure 4 in which colors represent individual elements. As is clearly seen, the elemental species coming from the pre-prototype material, like Ti, V, and Al, are located on the lateral surface of the spikes (as is O, which is not shown) while Ca and P are distributed only as deposits in the inter-spike space. This phenomenon could be explained by considering the distance between the spikes. It is most likely caused by insufficient room between the spikes. The unwanted result of the CaP ECDV=const surface modification of the PSc200 MSC-Scaffold pre-prototypes led to the decision to abandon further research using this geometrical variant of MSC-Scaffold pre-prototype.  For the PSc350 MSC-Scaffold pre-prototypes modified using VECD values ranging between −9 to −5.50 V, the weight increase of the surface coating was low (less than 1 mg) and the Ca/P ratios determined in the deposited surface coatings did not correspond to the Ca/P values characteristic for CaPs. A significant increase in weight (about 5 mg) was found when applying VECDvalues ranging between −5.25 to −4.75V. For the ECD process carried out using VECD values above −4.50 V, a slight weight increase was observed (approximately 0.50-0.75 mg). Unfortunately, the Ca/P ratio in the deposited surface coating did not correspond to the characteristic Ca/P values for native osseous For the P Sc350 MSC-Scaffold pre-prototypes modified using V ECD values ranging between −9 to −5.50 V, the weight increase of the surface coating was low (less than 1 mg) and the Ca/P ratios determined in the deposited surface coatings did not correspond to the Ca/P values characteristic for CaPs. A significant increase in weight (about 5 mg) was found when applying V ECD values ranging between −5.25 to −4.75V. For the ECD process carried out using V ECD values above −4.50 V, a slight weight increase was observed (approximately 0.50-0.75 mg). Unfortunately, the Ca/P ratio in the deposited surface coating did not correspond to the characteristic Ca/P values for native osseous CaPs. EDS analysis of all the P Sc350 MSC-Scaffold pre-prototypes modified using V ECD values of −5.25, −5.00 and −4.75 V confirmed the presence of CaPs having the Ca/P ratios consistent with the native osseous CaPs). Therefore, V ECD values from −5.25 to −4.75V can be recommended as the most suitable conditions for the CaP ECD V=const surface modification of the P Sc350 MSC-Scaffold pre-prototypes. Figure 5 shows the dependence of the average weight increase of the P Sc350 MSC-Scaffold pre-prototypes modified using the V ECD values of −5.25, −5.00 and −4.75 V. The dependence was determined both for the MSC-Scaffold pre-prototypes that underwent the AAT pretreatment and those that did not.  Figure 5 shows the dependence of the average weight increase of the PSc350 MSC-Scaffold pre-prototypes modified using the VECD values of −5.25, −5.00 and −4.75 V. The dependence was determined both for the MSC-Scaffold pre-prototypes that underwent the AAT pretreatment and those that did not. In both cases, the highest average weight increase for the modified PSc350 MSC-Scaffold pre-prototypes was obtained for VECD = −5.00 V. It can be clearly seen from Figure 4 that AAT pretreatment impacts the weight increase of the deposited CaP coating (by 44% for VECD = −5.25 V, by 9% for VECD = −5.00 V and by 15% for VECD = −4.75 V). Figure 6 shows PSc350SEM images of the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by a one hour ECDV=const process carried out using VECD values of −5.25, −5.00, and −4.75 V, followed by 48h incubation in SBF, without AAT pretreatment (Figure 6a-c) and with AAT pretreatment (Figure 6d In both cases, the highest average weight increase for the modified P Sc350 MSC-Scaffold pre-prototypes was obtained for V ECD = −5.00 V. It can be clearly seen from Figure 4 that AAT pretreatment impacts the weight increase of the deposited CaP coating (by 44% for V ECD = −5.25 V, by 9% for V ECD = −5.00 V and by 15% for V ECD = −4.75 V). Figure 6 shows P Sc350 SEM images of the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by a one hour ECD V=const process carried out using V ECD values of −5.25, −5.00, and −4.75 V, followed by 48 h incubation in SBF, without AAT pretreatment (Figure 6a-c) and with AAT pretreatment (Figure 6d-f).  Figure 5 shows the dependence of the average weight increase of the PSc350 MSC-Scaffold pre-prototypes modified using the VECD values of −5.25, −5.00 and −4.75 V. The dependence was determined both for the MSC-Scaffold pre-prototypes that underwent the AAT pretreatment and those that did not. In both cases, the highest average weight increase for the modified PSc350 MSC-Scaffold pre-prototypes was obtained for VECD = −5.00 V. It can be clearly seen from Figure 4 that AAT pretreatment impacts the weight increase of the deposited CaP coating (by 44% for VECD = −5.25 V, by 9% for VECD = −5.00 V and by 15% for VECD = −4.75 V). Figure 6 shows PSc350SEM images of the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by a one hour ECDV=const process carried out using VECD values of −5.25, −5.00, and −4.75 V, followed by 48h incubation in SBF, without AAT pretreatment (Figure 6a-c) and with AAT pretreatment (Figure 6d SEM analysis of the microstructure of the lateral spike surfaces shows that the CaP coating obtained during the ECDV=const process carried out without AAT pretreatment is non-uniform and seems to be unstable (the surface is not consistent). For MSC-Scaffold pre-prototypes modified using −5.25 V, most of the lateral spike surfaces remained uncoated in their medial part. The coating was deposited mostly on the upper part of the spikes. For the remaining pre-prototypes (modified using the VECD values of −5.00 and −4.75 V) the entire lateral surface of spikes was CaP coated, but numerous micro-cracks, especially for VECD = −5.00 V, were noted.

The Influence of the AAT Pretreatment
As can be clearly seen in the SEM images presented in Figure 6d-f, applying an AAT pretreatment has increased the coverage degree of the spike surfaces and the uniformity (no micro-cracks appear on the spike surfaces) of the produced CaP coatings for all VECD values of the ECDV=const process. Plate-like and needle-like shaped CaP crystals appear on the lateral surfaces of the MSC-Scaffold pre-prototypes. In particular, a significant accumulation of such crystals can be observed in the upper part of the MSC-Scaffold's spikes.
From the EDS analysis, the molar ratios of calcium to phosphorous on the lateral spike surfaces were 1.58-1.74, which is consistent with the values of native osseous CaP. The graph in Figure 7 shows the coverage degree of lateral spike surfaces of PSc350 MSC-Scaffold pre-prototypes that underwent one hour of ECDV=const process carried out using VECD values of −5.25, −5.00 and −4.75 V followed by 48h immersion SBF, with and without AAT pretreatment as a function of the applied VECD during the ECDV=const process. Figure 8 shows examples of the EDS chemical mapping for the lateral spike surfaces of the MSC-Scaffold pre-prototypes. The examples correspond with the results presented in Figure 7. SEM analysis of the microstructure of the lateral spike surfaces shows that the CaP coating obtained during the ECD V=const process carried out without AAT pretreatment is non-uniform and seems to be unstable (the surface is not consistent). For MSC-Scaffold pre-prototypes modified using −5.25 V, most of the lateral spike surfaces remained uncoated in their medial part. The coating was deposited mostly on the upper part of the spikes. For the remaining pre-prototypes (modified using the V ECD values of −5.00 and −4.75 V) the entire lateral surface of spikes was CaP coated, but numerous micro-cracks, especially for V ECD = −5.00 V, were noted.
As can be clearly seen in the SEM images presented in Figure 6d-f, applying an AAT pretreatment has increased the coverage degree of the spike surfaces and the uniformity (no micro-cracks appear on the spike surfaces) of the produced CaP coatings for all V ECD values of the ECD V=const process. Plate-like and needle-like shaped CaP crystals appear on the lateral surfaces of the MSC-Scaffold pre-prototypes. In particular, a significant accumulation of such crystals can be observed in the upper part of the MSC-Scaffold's spikes.
From the EDS analysis, the molar ratios of calcium to phosphorous on the lateral spike surfaces were 1.58-1.74, which is consistent with the values of native osseous CaP. The graph in Figure 7 shows the coverage degree of lateral spike surfaces of P Sc350 MSC-Scaffold pre-prototypes that underwent one hour of ECD V=const process carried out using V ECD values of −5.25, −5.00 and −4.75 V followed by 48 h immersion SBF, with and without AAT pretreatment as a function of the applied V ECD during the ECD V=const process. Figure 8 shows examples of the EDS chemical mapping for the lateral spike surfaces of the MSC-Scaffold pre-prototypes. The examples correspond with the results presented in Figure 7.
The EDS mapping results for the modified lateral spike surfaces and the quantitative analysis performed using ImageJ show that the greatest lateral spike surface coverage degree was obtained for the P Sc 350 MSC-Scaffold pre-prototypes modified at V ECD = −5.00 V (average 68±6%). For other V ECD values, the coverage degree of the lateral spike surfaces was half the size (33±5-35±5%). Applying of AAT pretreatment increases the coverage degree of the lateral spike surfaces (40% for V ECD = −5.25 V, 14% for V ECD = −5.00 V, and 100% for V ECD = −4.75 V).  The EDS mapping results for the modified lateral spike surfaces and the quantitative analysis performed using ImageJ show that the greatest lateral spike surface coverage degree was obtained  The EDS mapping results for the modified lateral spike surfaces and the quantitative analysis performed using ImageJ show that the greatest lateral spike surface coverage degree was obtained

XRD Analysis
To confirm the crystalline form of CaP was deposited on the surface of MSC-Scaffold pre-prototypes due to electrochemical treatment using V ECD = −5.00 V, an XRD analysis was performed. The results are shown in Figure 9. for the PSc350 MSC-Scaffold pre-prototypes modified at VECD = −5.00 V (average 68±6%). For other VECD values, the coverage degree of the lateral spike surfaces was half the size (33±5-35±5%). Applying of AAT pretreatment increases the coverage degree of the lateral spike surfaces (40% for VECD = −5.25 V, 14% for VECD = −5.00 V, and 100% for VECD = −4.75 V).

XRD Analysis
To confirm the crystalline form of CaP was deposited on the surface of MSC-Scaffold pre-prototypes due to electrochemical treatment using VECD = −5.00 V, an XRD analysis was performed. The results are shown in Figure 9. As one can see, the obtained CaP coating is multiphasic and there are identified peaks attributed to CaP phases like octacalcium phosphate, Ca8H2(PO4)6•5H2O (PDF2# 00-026-1056), calcium metaphosphate, Ca(PO3)2 (PDF2# 00-003-0348), and monocalcium phosphate monohydrate, Ca(H2PO4)2•H2O (PDF2# 00-003-0284). Apart from this, the impurity phases from the SBF solution were found on the surface sodium chloride, NaCl (PDF2# 01-077-2064), and sodium hydrogen carbonate, NaHCO3 (PDF2# 00-021-119). The results of the XRD investigation showed that, during the combined ECD process conducted under the determined conditions, CaP biomineral coating is produced on the lateral surface of spikes of the MSC-Scaffold.

Discussion
In this study, the experimental CaP modification of the bone-contacting surfaces of MSC-Scaffold pre-prototypes was undertaken in search of the suitable range of conditions for CaP deposition in the ECDV=constprocess. Since the MSC-Scaffold prototype, as the original concept of a multi-spiked (needle-palisade) fixation of RA endoprostheses components, was developed within the frames of two of our research projects, its most suitable geometrical properties evolved based on the findings of bioengineering research in relation to that primarily suggested in the patented version [3][4][5]. Additionally, the conditions ECD modifications have changed accordingly. Attempts to modify the bone-contacting surfaces of MSC-Scaffold prototypes initially undertaken at constant current densities were satisfactory [52], i.e., it is possible to control the deposition of CaPs on the bone-contacting surfaces of MSC-Scaffold pre-prototypes by adjusting the current density [52].Significant enhancement of the osteoinduction/osseointegration potential of the MSC-Scaffold prototype was confirmed in pilot experimental studies in animal models and in osteoblast cultures [53]. After the structural-geometric functionalization of the additively manufactured prototype MSC-Scaffold, we observed that the results of CaP modifications of the bone-contacting surfaces of MSC-Scaffold prototypes carried out during the potentiostatic process (ECDV=const) showed much higher repeatability compared to those of the galvanostatic process (ECDj=const). As one can see, the obtained CaP coating is multiphasic and there are identified peaks attributed to CaP phases like octacalcium phosphate, Ca 8 H 2 (PO 4 ) 6 ·5H 2 O (PDF2# 00-026-1056), calcium metaphosphate, Ca(PO 3 ) 2 (PDF2# 00-003-0348), and monocalcium phosphate monohydrate, Ca(H 2 PO 4 ) 2 ·H 2 O (PDF2# 00-003-0284). Apart from this, the impurity phases from the SBF solution were found on the surface sodium chloride, NaCl (PDF2# 01-077-2064), and sodium hydrogen carbonate, NaHCO 3 (PDF2# 00-021-119). The results of the XRD investigation showed that, during the combined ECD process conducted under the determined conditions, CaP biomineral coating is produced on the lateral surface of spikes of the MSC-Scaffold.

Discussion
In this study, the experimental CaP modification of the bone-contacting surfaces of MSC-Scaffold pre-prototypes was undertaken in search of the suitable range of conditions for CaP deposition in the ECD V=const process. Since the MSC-Scaffold prototype, as the original concept of a multi-spiked (needle-palisade) fixation of RA endoprostheses components, was developed within the frames of two of our research projects, its most suitable geometrical properties evolved based on the findings of bioengineering research in relation to that primarily suggested in the patented version [3][4][5]. Additionally, the conditions ECD modifications have changed accordingly. Attempts to modify the bone-contacting surfaces of MSC-Scaffold prototypes initially undertaken at constant current densities were satisfactory [52], i.e., it is possible to control the deposition of CaPs on the bone-contacting surfaces of MSC-Scaffold pre-prototypes by adjusting the current density [52].Significant enhancement of the osteoinduction/osseointegration potential of the MSC-Scaffold prototype was confirmed in pilot experimental studies in animal models and in osteoblast cultures [53]. After the structural-geometric functionalization of the additively manufactured prototype MSC-Scaffold, we observed that the results of CaP modifications of the bone-contacting surfaces of MSC-Scaffold prototypes carried out during the potentiostatic process (ECD V=const ) showed much higher repeatability compared to those of the galvanostatic process (ECD j=const ).
Experimental CaP modification of the MSC-Scaffold pre-prototypes was carried out in two steps. In the first step, the purpose was to determine the most suitable range of conditions for the ECD V=const process. V ECD values from the −9 to −3V range were applied. This stage of investigations showed that the suitable conditions for the ECD V=const process of CaP modification of complex-shaped bone-contacting surfaces of the MSC-Scaffold prototype are strongly influenced by the geometrical features of the scaffold prototype, i.e., by the distance between the spikes. In the case of insufficient room between the MSC-Scaffold's spikes, the CaP deposits are found between the spikes instead of on their lateral surface. Hence, the P Sc200 variant of the MSC-Scaffold pre-prototype was excluded from further research. Based on the characterization of the coating's physiochemical properties-in terms of structural (EDS) and morphological (SEM) properties, and the weight increase of the deposits-the V ECD range from −5.25 to −4.75V was determined to provide the expected CaP modification of the bone-contacting surfaces of the P Sc350 variant of the MSC-Scaffold pre-prototype.
In the second step, the influence of AAT pretreatment was examined by applying the previously determined range of V ECD values that achieve the native biomineral Ca/P ratio in coatings on the lateral surface of the MSC-Scaffold pre-prototypes. The investigation procedure applied in the first step was extended to include EDS surface mapping and quantitative analysis of crystalline phases (XRD). The enhancement of the coverage degree of the lateral spike surfaces and the coverage uniformity were ascertained. AAT pretreatment prevents micro-crack formation on the bone-contacting surfaces of the MSC-Scaffold and also affects the increase of the spikes' lateral surface coverage. The Ca/P ratios of deposits on the lateral spike surfaces in all modified MSC-Scaffold pre-prototypes are consistent with the Ca/P ratios of native osseous CaPs, and plate-like and needle-like CaP crystals appeared on the bone-contacting surface of the MSC-Scaffold pre-prototypes undergoing the AAT pretreatment.
The best overall results for CaP modification of the bone-contacting surfaces of the MSC-Scaffold pre-prototypes were obtained for the V ECD = −5.00 V-the native biomineral Ca/P ratio of deposits (i.e., the closest values to the Ca/P ratio native osseous CaP) was achieved, as well as the highest average mass growth of the coating and the highest coverage degree of spikes' lateral surface (even in the case of the MSC-Scaffold prototypes without AAT pretreatment). The numerous micro-cracks observed on the MSC-Scaffold pre-prototypes CaP modified at V ECD = −5.00V were prevented by applying the AAT pretreatment, finally providing the highest uniformity in comparison to the other V ECD values.

Conclusions
The effect of CaP ECD deposition on the MSC-Scaffold prototype is conditioned not only by the appropriate choice of electric parameter values of the ECD process but also on the geometrical features (distance between the spikes) of the MSC-Scaffold. Of the two examined design variants, the distance between spike bases of 200 µm (P Sc200 ) and 350 µm (P Sc350 ), the P Sc200 MSC-Scaffold variant appeared to be inappropriate to be CaP modified in the ECD V=const process.
Based on the SEM and EDS studies of deposited CaP coatings together with the measurements of the weight increase for the P Sc350 MSC-Scaffold pre-prototypes, the most suitable V ECD values for the ECD V=const process were from −5.25 to −4.75 V and the best results for CaP modification of their bone-contacting surfaces was obtained at V ECD = −5.00 V; the native biomineral Ca/P ratio of coatings was achieved for all the V ECD values used.
ECD combined with AAT pretreatment prevents micro-crack formation on the bone-contacting surfaces of the MSC-Scaffold prototype and increases the spikes' lateral surface coverage; the best results for the CaP modification of the bone-contacting surfaces was obtained at V ECD = −5.00 V.
Thus, the biomimetic MSC-Scaffold prototype with desired biomineral coating of native Ca/P ratio on bone-contacting surfaces was obtained for a new kind of entirely non-cemented resurfacing arthroplasty endoprostheses.