**3. Results**

Before cutting the test specimen, each plate was checked for the existence of Alpha Case, a critical surface feature, which can develop during the cooling phase of the casting process due to the presence of oxygen. While thin layers can be removed by etching, generally avoiding the formation of Alpha Case during the casting process is the preferred solution with respect to cost-e ffective part production. Figure 3 shows typical analyses of cast part samples before (Figure 3A,C) and after (Figure 3B,D) optimization of the employed centrifugal casting process, indicating that Alpha Case formation could be completely prevented in the test samples used for further analyses (Figure 3B,D).

**Figure 3.** Microstructure analysis (Light Microscopy 100× magnification; Micro-Hardness) ( **A**,**C**) Microstructure before process optimization, thickness of Alpha Case Layer critical (>15 μm); (**B**) and (**D**) Microstructure after process optimization, no Alpha Case detectable (Hardness Vickers, Load: HV0.10, Obj. 50×).

The chemical composition of the test specimen was checked by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analysis according to DIN 51008-2/DIN 510009. ICP-OES is an elemental analysis method to determine sample compositions on trace-level. Samples are water dissolved and conducted through a nebulizer into a spray chamber. The resulting aerosol is lead through an argonized plasma chamber operating at around 6000 to 7000 K. The aerosol takes up thermal energy and atomization, and ionization takes place. Electrons reach a higher state for a short amount

of time and eventually drop back to ground level energy. While dropping back, energy is liberated as light waves (photons). For each element, the wavelength of the emitted light is characteristic and used to identify the elements present in the sample. Information regarding the content of an element is provided by the measured intensity of the detected wave lengths. Based on the machine calibration, these values are used to calculate the concentration of elements contained in the sample.

Representative results are shown in Table 2. The element amounts were within the specified boundaries and the results show a typical decrease of Al and an increase of elements such as Cu, Fe and Ti, respectively. This e ffect commonly occurs when a melting alloy containing Al is used at high temperatures. However, this e ffect is minimal for the Leicomelt 5 TP device, as shown by the results.


**Table 2.** Element concentration as specified for Ti6Al4V ELI and measured for the alloy material before and after the melting+casting+HIP process chain. Tests were conducted by Elektrowerk Weisweiler GmbH (Eschweiler-Weisweiler, Germany). Results are shown in weight percent (wt.%).

#### *3.1. Surface Characteristics and SEM Analysis*

The biocompatibility of medical implants is determined by several factors. Although the reference and cast specimens used in this study were made of the same titanium alloy and had a comparable major chemical composition as mentioned above, e ffects on the cell adhesion, proliferation, and di fferentiation of human osteoblasts could be influenced by the respective surface profile. Therefore, surface micro-topography was characterized.

When analyzing the average roughness Ra and Rz by using standard procedures, no significant di fferences were detected. For the reference surfaces, the mean value of Ra was 5.1 μm (SD 0.44 μm), while a Rz of 29.78 μm (SD 1.71 μm) was measured. For the cast samples, the mean value determined for Ra was 4.55 μm (SD 0.14 μm) and Rz ranged from 28.05 μm (SD 2.18 μm). In ion implanted surfaces, samples with implanted Ca-ions showed mean values of Ra 4.4.5 μm (SD 0.43 μm) and Rz 27.20 μm (2.53 μm). In cast specimens with implanted P-ions, Ra was measured with 4.86 μm (SD 0.45 μm), and Rz 29.58 μm (SD 2.07 μm) was detectable.

By using scanning electron microscopy, we could further show that the surfaces of both types of specimen were similar and that the seeded human osteoblasts could adhere and cover the surface after several days, as shown in Figure 4. Additionally, no di fferent seeding behavior was observed after the ion beam implantation of calcium or phosphorus ions into the surface. The osteoblasts were comparable in terms of their cellular morphology and size.

(**e**) (**f**) 

**Figure 4.** SEM images of cell seeded surfaces. On all shown pictures, primary human osteoblasts were cultivated for three days under standard conditions. Reference material (**a**) 200 k and (**b**) 725 k (magnification). Cast specimens (**c**) 200 k and (**d**) 625 k (magnification). Cast specimens with ion beam implantation of Ca (ion density 1 × 10−<sup>16</sup> cm2, energy level 30 keV) ((**e**) 200 k and (**f**) 800 k magnification). Cast discs with ion beam implantation of P (ion density 1 × 10−<sup>16</sup> cm2, energy level 30 keV) ((**g**) 200 k and (**h**) 800 k magnification). Scanning electron microscopy was performed using a Hitachi S-5200 (Tokyo, Japan). Pictures were taken from [18] with permission.

(**g**) (**h**)

It can be summarized that conspicuous changes in cell morphology could be detected on none of the investigated casting surfaces. Therefore, with regard to cell adhesion and cell spreading, an equivalence between centrifugal casting and the current reference surfaces can be assumed. Additionally, the ion beam implantation of calcium or phosphorus ions did not obviously affect the respective cellular response.

#### *3.2. Cell Adhesion and Proliferation*

In terms of biocompatibility, the cell adhesion and proliferation/viability measured by MTT-assay can be used as a well-established tool for characterizing cellular interaction with biomaterials. As shown in Figure 5, on all tested surfaces the human osteoblasts could adhere in equal amounts after 24 h. No statistically significant difference was observed between the reference material and the specimen produced by centrifugal casting. In addition, after cultivation for 7 days, no significant difference could be identified. Regarding the time of cultivation, a significant difference between day 1 and day 7 was detectable for the reference (REF) and the cast material (CAST). The ion implantation of P led to a nearly significant increase from day 1 to day 7 (CAST+P; *p* = 0.055). After the ion implantation of Ca, the mean cell number at day 7 was lowest compared with the other groups, and no significant increase compared with day 1 could be calculated (*p* = 0.24).

In summary, the type of production of the discs did not inhibit the adhesion and proliferation of human osteoblastic cells. All tested groups showed an increase in cell numbers over time.

In addition, by comparing all tested groups of material surfaces, no statistically significant difference was observed (*p* > 0.99).

#### *3.3. Optimized Centrifugal Casting Did Not Impair Osteogenic Di*ff*erentiation*

In the presented work, we also investigated whether osteogenic differentiation of human osteoblasts was influenced by cultivation on cast surfaces or reference material. Target genes for the analysis of osteogenic differentiation markers on gene expression levels were RUNX2, SP7, COL1a1, ALP and BGLAP, and markers for bone homoeostasis were OPG, RANKL, and CASP3 for the induction of cell apoptosis, as shown in Figure 6.

**Figure 5.** Cell proliferation tested by MTT-assay. Besides the comparison of reference material (REF) and cast specimens (CAST), the influence of beamline-implanted Ca- or P-ions into surfaces on cytotoxicity/proliferation was also tested. The proliferation of osteoblasts on the surfaces was investigated on day 1 and day 7 (*n* = 7 each). The quotients of OD 570–650 nm were measured, and respective values are shown as scatter plots with Median and interquartile ranges. Influences of time as well as surfaces were analyzed by two-way ANOVA and Bonferroni post hoc tests for statistical significance. The significance level was set to *p* ≤ 0.05. OD: Optical Density; Ca: Ca-ion; P: P-ion; Implantation dose 1 × 10−<sup>16</sup> cm2, implantation energy 30 keV.

The Runt-related transcription factor 2 (RUNX2) as a key regulator of osteoblast di fferentiation was expressed in equal amounts on all tested surfaces. Additionally, the expression of transcription factor SP7 (Osterix), a regulator of osteogenic di fferentiation that enhances the e ffect of RUNX2, was not modulated by the di fferent surfaces. So, the main transcriptional regulators of osteogenic di fferentiation were not influenced by the centrifugal cast materials.

In addition, later relevant gene expressions of the osteoblastic phenotype, like COL1a1 (collagen type I, the essential collagenous component for building bone matrix), ALP (Alkaline phosphatase, needed for mineralization of bone matrix), and BGLAP (Osteocalcin, needed to build hydroxyapatite crystals within the bone matrix), were not significantly modulated over the tested time period of 7 days.

With respect to bone resorption, the tested marker genes OPG (inhibitor of bone resorption) and RANKL (stimulator of bone resorption) showed no significant di fferences between the CAST and reference specimen (OPG *p* = 0.22, RANKL *p* > 0.99). However, the implantation of Ca- (*p* = 0.015) or P-ions (*p* = 0.005) significantly reduced the gene expression level for OPG. The expression for RANKL was not modified for any group (*p* > 0.99).

The induction of apoptosis to the osteoblasts was tested by CASP3 analysis. Caspase 3 is a key regulator for cell death. No significant di fference (*p* = 0.999) on gene expression level was observed after 7 days of cultivation on the tested specimen.

In summary, on gene expression level, the spin-cast and the reference material did not show significant di fferences. In terms of cast material, the additional Ca-/P-ion implantation significantly reduced the gene expression level of OPG.

We further analyzed the ongoing di fferentiation process of protein and on mineralization levels by quantification of C-terminal collagen type I propeptide (CICP) as a measure of collagen type I synthesis and Osteoprotegerin (OPG), as well as Alizarin red S staining, as shown in Figure 7. All data on protein expression were normalized to the respective MTT values as a measure of cell number.

(**b**) Markers for differentiation COL1a1, ALP and BGLAP.

**Figure 6.** *Cont.*

(**c**) Marker for bone homeostasis OPG and RANKL.

(**d**) Marker for apoptotic process CASP3.

**Figure 6.** Gene expression analysis of primary human osteoblasts after cultivation for 7 days on a cast Ti6Al4V specimen without and with Ca- or P-ion implantation as well as a reference Ti6Al4V-specimen. Diagramed are scatterplots showing the interquartile range of the calculated ΔΔCt gene expression. (**a**) Shows results of regulators of the osteogenic phenotype. While RUNX2 (Runt-related transcription factor 2) is a key regulator in osteoblast differentiation, SP7 (Osterix) is a regulator of osteogenic differentiation and enhances the effect of RUNX2. (**b**) Presents the gene expression of differentiated osteoblasts. COL1a1: collagen type I is the major structural protein of bone. ALP: alkaline phosphatase is needed for mineralization of bone matrix and BGLAP: osteocalcin is needed to build hydroxyapatite crystals within the bone matrix. (**c**) Markers for bone homeostasis. OPG: osteoprotegerin is a decoy receptor for RANKL (receptor activator of nuclear factor kappa-B ligand), while RANKL normally binds to RANK in order to stimulate bone resorption by osteoclasts. (**d**) Marker for the apoptotic process. CASP3: caspase 3 is a predominant caspase and regulates cell apoptosis. Gene expression was normalized in osteoblasts cultivated on cast surfaces without ion implantation. All gene expression data were normalized internal to HPRT1 as a housekeeping gene. Statistical analysis by one-way ANOVA with Kruskal–Wallis and Dunn's post hoc test. The significance level was set to *p* < 0.05 (\*). Ca: Ca-ion; P: P-ion. Ion dose 1 × 10−<sup>16</sup> cm2, implantation energy 30 keV; *n* = 7.

The CICP concentration in the cell culture supernatant, reflecting the amount of procollagen type I biosynthesis by the osteoblasts seeded on cast surfaces, declined from day 3 (730.2 pg/mL) until day 7 (272.4 pg/mL, *p* < 0.07), indicating a negative feedback mechanism of pericellular collagen type I accumulation (Figure 7a). Osteoblasts seeded on reference surfaces produced, according to the current standard method, similar results (day 3: 912 pg/mL; day 7: 240 pg/mL) for CICP-concentration, although the decline was statistically significant in this case. In the cell culture supernatant of Ca-ion implanted casting surfaces, a CICP level of 706.8 pg/mL could be observed, while on day 7 the secretion decreased 1.7-fold to 415 pg/mL (*p* > 0.99). The amount of CICP in the cell culture supernatant of P-ion implanted casting surfaces was somewhat lower compared with Ca-ion implanted surfaces, although the di fference was not significant. Over time, the median CICP of 611.5 pg/mL decreased by a factor of 2.5 to 244 pg/mL (*p* = 0.09) on day 7. In the cell culture supernatant of the reference surfaces, a 3.8-fold significant decrease in CICP to 239.5 pg/mL was observed on day 7 (*p* = 0.005). Overall, no significant di fference in type I procollagen production of osteoblasts on cast surfaces without or with ion implantation could be determined compared with the reference surfaces.

OPG served as a bone turnover marker and is produced by osteoblasts to counteract bone resorption by osteoclasts. OPG is a decoy receptor for RANKL in the RANK/RANKL/OPG axis and is essential to bone metabolism. On the non-ion-implanted casting surfaces, normalized to MTT-values, an OPG release of 2673 pg/mL could be measured on day 7 (Figure 7b). Furthermore, it was shown that the OPG concentration in the cell culture supernatant of Ca-ion implanted cast surfaces was 3840 pg/mL and thus 1.4 times higher than P-ions implanted surfaces (2779 pg/mL). The OPG secretion of the osteoblasts, cultivated on reference surfaces, was similar to cast surfaces without and with P-ion implanted surfaces (median of 2833 pg/mL). Overall, there were no significant di fferences for OPG release of human osteoblastic cells on the surfaces (*p* < 0.99).

Figure 7c presents data of an Alizarin red S staining of osteoblast-like cells cultivated on cast material and on reference material shown after 14 days. The median Alizarin red S concentration of osteoblasts on casting surfaces was 139.0 μg/mL. For reference discs, a median Alizarin red S concentration of 152.3 μg/mL was measured. There was no significant di fference between reference and cast surfaces (*p* = 0.88). The Alizarin red S values of the cells cultivated on the Ca-ion implanted surfaces was significantly reduced by 27.8% compared with non-modified cast surfaces (*p* = 0.02). For P-ion implantation in casting material, the values were also somewhat lower compared with non-ion-implanted material but not significantly di fferent (*p* = 0.16). Neither comparing CAST-Ca (*p* = 0.15) nor CAST-P (*p* = 0.30) with the reference material showed statistically significant di fferences.

**Figure 7.** *Cont.*

**Figure 7.** Detection of in vitro mineralization and protein secretion. (**a**) CICP Elisa for collagen type I synthesis after osteoblast cultivation for 3 and 7 days. Influence of different surfaces on collagen type I synthesis was tested by a two-way ANOVA with Bonferroni post hoc test. (**b**) OPG Elisa results of osteoblast cultures after 7 days cultivation. Data were analyzed by using Kruskal–Wallis and Dunn's post hoc tests for statistical significance. (**c**) Alizarin red S staining was used to analyze the mineralization of osteoblasts' seeded specimens and the effect of Ca- or P-Ion coated surfaces after 14 days. Statistical analysis was done by a one-way ANOVA with a Tukey post hoc test. The significance level was set to *p* ≤ 0.05. Values are shown as scatter plots with Median and interquartile range. Ca-ion and P-ion implantation by ion dose 1 × 10−<sup>16</sup> cm2, implantation energy 30 keV; *n* = 7.

In summary, this cell biologic analyses showed that there was no significant difference between the examined cast surfaces and the reference surfaces with regard to procollagen type I and OPG secretion as well as in vitro mineralization. The additional ion implantation of Ca or P did not show any supportive effect either.
