**1. Introduction**

Nowadays, there is high demand for cost-e ffective knee joint endoprosthesis in orthopedic arthroplasty. The currently used respective Gold standard material for cemented endoprosthesis, Co28Cr6Mo, however, is in discussion in terms of biocompatibility and longevity because of its cobalt content. Ti6Al4V is the most frequently used titanium-alloy for uncemented orthopedic endoprosthesis

implantation, but fabrication of standard 3-dimensional structures for joint arthroplasty involves cost intensive usage of machining blanks and milling machines to remove projecting titanium parts [1].

Therefore, a cost-e ffective titanium casting process could achieve improvement in several ways. It could lead to the partial replacement of cobalt-containing implants and avoid the use of bone cement in these cases based on superior features of osseointegration. However, because of the high melting point of titanium and its unfavorable fluidity and reactivity in a molten state, this fabrication procedure is not routinely used so far [2,3].

As described previously, an optimized centrifugal casting process, including casting and cooling conditions, crucible and mold material, and enhanced heat treatment facilitates, was used for the manufacturing of near net-shape titanium-based implants (Figure 1 is an illustration of a centrifugal precision casting device from Michels, Aachen, Germany) [4]. In the applied casting process on the Leicomelt 5 TP casting device, the solid metal alloy is placed in a cold wall crucible. This type of crucible consists of a ring-shaped wall of water-cooled palisades made from copper. A magnetic alternating field is induced by the application of current to an induction coil surrounding the palisade package. This field generates heat in the alloy material due to ohmic losses, eventually melting the solid alloy. Melting and subsequent casting is carried out under an inert gas/vacuum atmosphere. The liquid metal is poured by tilting the crucible through a separation valve between the melting and casting chamber into a heating box containing the casting setup consisting of sprue, melt distributor and ceramic shell mold. The heat box and casting setup are fixed on a rotating table inside the casting chamber. Prior to the pouring of the melt, the casting the table is set into a rotation of up to 400 RPM, creating rotational forces on the liquid metal as it enters the casting setup. Following the applied force, the melt flows into the ceramic shell mold, thus filling it within 1.5 s. The cooled down metal is manually freed from the ceramic shell mold, and the in vitro samples are cut by a water jet.

**Figure 1.** Principal centrifugal casting layout (Leicomelt TP5) (**left**); cast part wax model providing 4 rectangular plates, water jet cutting lines in red (**center**); water-jet cut of in vitro test specimen (**right**). Discs were 14 mm in diameter and 2 mm in height.

Besides the complex manufacturing process of titanium-based implants, it is necessary to focus on their biological aspects. Stability after uncemented implantation into bone depends on a proper interaction between the material and the cells of the surrounding tissue, especially osteoblasts [5]. While pure titanium or titanium-based alloys like Ti6AL4V are usually regarded as having excellent mechanical and biocompatibility properties, several organic and inorganic surface modifications were used to further enhance osseointegration [6]. With respect to inorganic components, besides surface modification by the addition of hydroxyapatite, complex 3-dimensional structures were generated by plasma treatment in order to deposit calcium or phosphorus ions onto the surfaces [7,8]. Since delamination of hydroxyapatite coatings with negative long-term e ffects has been reported [8,9], ion implantation of calcium and phosphorus into the material surface has been tested, encouraging the formation of calcium phosphate precipitates [10,11]

The alloy surface should maintain the cell adhesion, proliferation and di fferentiation processes of osteoblasts, which are dependent on biochemical, topographical and biomechanical parameters. One example is the increasing response of di fferent cells to materials using nano topography by supporting respective adhesion and proliferation [12,13]. With respect to osseointegration, the material should not decrease the expression of necessary osteogenic di fferentiation factors like RUNX2 (Runt-related transcription factor 2) or the synthesis of collagen type I, which is essential for extracellular matrix synthesis [14].

The aim of this in vitro study was to investigate the possibilities of using machine blanks and milling machines to transfer a medical grade process of knee endoprosthesis production into a more cost-e ffective, less material consuming optimized centrifugal casting process, without worsening the biological e ffects on primary human osteoblasts. We used a modified manufacturing technique for the production of near net-shape precision centrifugal castings of Ti6Al4V, representing an alloy widely used for medical treatment. In addition, a calcium and phosphorus ion beam implantation into the implant's surface was tested to see if it modified the biological outcome.

#### **2. Materials and Methods**

An overview of the experimental setup is given in Figure 2. Besides the used test specimen, the cell biological tests are illustrated.

**Figure 2.** Scheme of the materials and methods used for in vitro testing. The medical grade alloy Ti6Al4V was processed under standard conditions (reference material; REF) or in the optimized centrifugal casting process (CAST). Some specimens were modified by ion beam insertion of Ca- or P-ions into the surface. Discs of 14 mm in diameter were seeded with human primary osteoblasts and underwent di fferent in vitro testing. Cell biologic analysis focused on cell proliferation, osteogenic di fferentiation, matrix mineralization, remodeling processes, and apoptosis.

MTT: colorimetric assay of cellular metabolic activity by reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan. Alizarin red S: staining of the calcified matrix that is synthesized by the osteoblasts. Quantitative real time PCR: gene expression analysis of di fferentiation markers. Osterix/SP7: a transcription factor that is highly conserved among bone-forming cells and responsible for osteogenic di fferentiation. RUNX2: Runt-related transcription factor 2 associated with osteoblast di fferentiation. COL1a1: collagen type I alpha 1 as one chain of collagen type I—the major structural protein of bone. CICP: type I C-terminal collagen propeptide. ALP: tissue non-specific Alkaline phosphatase. BGLAP: osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein, is a calcium binding protein. CASP3: caspase-3 plays an important role in cell apoptosis. OPG: osteoprotegerin, a protein produced by osteoblasts to counteract bone resorption by osteoclasts. RANKL: the receptor activator of nuclear factor kappa-B ligand, stimulator of bone resorption. OC: osteoclasts.

#### *2.1. Primary Human Osteoblasts*

Primary human osteoblasts were isolated from human bone specimens collected from routine joint replacement surgery with the consent of patients and after approval from the local ethics committee of the University of Ulm. Overall, samples from 7 donors were used. Cells were isolated and cultivated under standard conditions as described previously [15].

## *2.2. Test Specimen*

For the in vitro testing, discs of the respective materials with a diameter of 14 mm and a height of 2 mm were used. The reference test specimens (REF) consisted of machined, aluminum-oxide-blasted Ti6Al4V and were manufactured by Peter Brehm GmbH (Weisendorf, Germany). The spin-cast discs were manufactured by the optimized centrifugal precision-casting technique, which was developed in cooperation between the independent research facility Access e. V. (Aachen, Germany) and the implant manufacturer, Peter Brehm GmbH (Weisendorf, Germany), as described earlier [6]. Following the casting process, the parts were heat HIPed (hot-isostatic pressed) according to Ti6Al4V standard procedures and parameters (1000 bar at 920 ◦C for 120 min). During the HIP procedure, by parallel application of temperature and pressure, the porosity in the cast part is e ffectively reduced, increasing the density of the cast material by significantly decreasing the volume fraction of possibly existing casting defects. The test specimens created by the melting cast procedure (CAST) were cut out of bigger sized plates to maintain the typical surface features generated by the casting process. Finally, the CAST discs were aluminum-oxide-blasted, using the same standard process as for the reference material [6].

Ion beam implantation was used to modify the properties of material surfaces [15]. Ca- or P-atoms were ionized and accelerated in electric fields and thus implanted into the surface of planar Ti6Al4V seals. The implantation was carried out with a conventional low-energy implant DANFYSIK 1050 (Danfsik, Taastrup, Denmark) at the Helmholtz-Zentrum Dresden-Rosssendorf (HZDR), as described previously for Cu- and Ag-ions [15]. The surfaces were implanted with an energy of 30 keV and a dose of 1 × 10−<sup>16</sup> cm<sup>2</sup> Ca- or P-ions.

#### *2.3. SEM Analysis of Cell Adhesion*

The surface structure of the discs and cell adhesion were investigated by using scanning electron microscopy Hitachi S-5200 (Hitachi, Tokyo, Japan) at the electron microscopy core facility of the University of Ulm, Germany. Specimens without cells were sputtered with gold-palladium (20 nm) under standard conditions. Specimens with cells were first fixed with 2.5% glutaraldehyde and 1% saccharose in 0.1 M of phosphate bu ffer before sputtering with gold-palladium.

#### *2.4. Molecular Biological Methods*

Gene expression analysis was performed with standard methods, as described earlier [16]. Human osteoblasts were seeded at a density of 20,000 cells per disc, which was placed in one well of a 24-well plate and covered with 1 ml of Dulbecco's Modified Eagle Medium (DMEM), 10% fetal calf serum (FCS), 100 μL penicillin/streptomycin solution, and 2 mM L-glutamine (all Biochrome, Berlin, Germany). For di fferentiation processes, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.2 mM L-Ascorbic acid (all Sigma-Aldrich, Steinheim, Germany) were added to the medium.

For normalization of the quantitative real-time polymerase chain reaction (PCR) results, a cell sample of osteoblasts before seeding on the titanium surfaces was used in all experiments. The cell culture supernatant was used for Enzyme Linked Immuno Sorbant Assay (Elisa). The cells were lysed by using the RNeasy kit from Qiagen (Qiagen, Hilden, Germany), following company instructions. Synthesis of cDNA was carried out with the Omniscript kit from Qiagen (Qiagen, Hilden, Germany) in accordance with the given manuals. Gene expression was analyzed using a TaqMan StepOne Plus

(Life Technologies, Darmstadt, Germany) and ready-to-use TaqMan probes, which are commercially available from Life Technologies (listed in Table 1). Amplifications were carried out using the TaqMan Fast Advanced Master Mix (Life Technologies, Darmstadt, Germany). Gene expressions were calculated with the ΔΔCt method and normalized to HPRT1 as a housekeeping gene [17].

**Table 1.** The TaqMan probes and targets used. Probes were designed and tested for specificity by Life Technologies (Darmstadt, Germany).


Gene and Gene Accession No. from GeneBank, NIH, TaqMan Assay ID given identification No from Life Technologies.
