**3. Results**

### *3.1. Determination of the Most Suitable Range of Conditions for the ECDV*=*constProcess*

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.

**Figure 2.** Mass increase of surface coating of the PSc200 and PSc350MSC-Scaffold pre-prototypes after the ECDV=const process as a function of the applied VECD value.

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.

**Figure 3.** Example SEM images showing the unwanted effect of CaP deposition in the inter-spike space of the PSc200 MSC-Scaffold pre-prototypes during the ECDV=const process; magnification: 30× and 300×.

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.

**Figure 4.** SEM and EDS mapping of the elemental species on the surface of the MSC-Scaffold pre-prototypes' spikes and deposits between the spikes (**a**) SEM morphology, (**b**) CaP map, (**c**) Ca map, (**d**) P map, (**e**) Ti map, (**f**) Al map and (**g**) V map.

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 CaPs. EDS analysis of all the PSc350 MSC-Scaffold pre-prototypes modified using VECD 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, VECD values from −5.25 to −4.75V can be recommended as the most suitable conditions for the CaP ECDV=constsurface modification of the PSc350 MSC-Scaffold pre-prototypes.

### *3.2. The Influence of the AAT Pretreatment*

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.

**Figure 5.** The average weight increase of the PSc350 MSC-Scaffold pre-prototypes as a function of applied VECD values for the MSC-Scaffold pre-prototypes with and without AAT pretreatment.

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 48 h incubation in SBF, without AAT pretreatment (Figure 6a–c) and with AAT pretreatment (Figure 6d–f).

**Figure 6.** *Cont*.

**Figure 6.** SEM 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: (**a**) −5.25 V, (**b**) −5.00 V and (**c**) −4.75 V, followed by 48 h incubation in SBF without AAT pretreatment, and, correspondingly, (**d**–**f**) with AAT pretreatment.

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.

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 48 h 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.

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 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).

**Figure 7.** The coverage degree of the lateral spike surfaces of the PSc350 MSC-Scaffold pre-prototypes after one hour ECDV=const carried out at VECD values of −5.25, −5.00 and −4.75 V followed by 48 h incubation in SBF, with and without AAT pretreatment as a function of the applied VECD.

**Figure 8.** Example results of the EDS mapping of the elemental species on the surface of the MSC-Scaffold pre-prototypes' spikes and deposits between the spikes: (**a**) SEM morphology, (**b**) CaP map, (**c**) Ca map, (**d**) P map, (**e**) Ti map, (**f**) Al map and (**g**) V map.
