**4. Discussion**

We aimed to show that an optimized spin casting process of Ti6Al4V, a frequently used titanium alloy for the production of hip endoprostheses for uncemented implantation, can generate material surface characteristics with comparable in vitro biocompatibility for primary human osteoblasts as the standard machined manufacturing process. Ion beam implantation of calcium or phosphorus following aluminum oxide blasting of the cast material, however, did not further improve the cell-biologic outcome in the dosage used.

With respect to material properties, standardized analysis did not reveal an indication of Alpha Case formation, confirming the positive e ffect of the optimized casting process with respect to metallurgical quality. Furthermore, chemical composition of the cast parts was within the specification. Therefore, no relevant reaction between melt and crucible or shell mold could be observed. Additionally, subsequent aluminum oxide blasting led to expected surface roughness with no significant di fference to the reference machined material.

In vivo bone response to cast titanium implants in the tibia metaphysis has been previously studied in a rabbit and a rat model [4,19]. Mohammadi et al. used cast pure titanium from which about 0.25 mm of the superficial layer was eliminated by machining to remove impurities created by the casting process [19]. Our recent publication on a rat model was based on the same optimized casting process of the titanium alloy Ti6Al4V without removal of the superficial layer before aluminum oxide blasting, as used in the present study [4]. In both animal models, no significant di fference in osseointegration after 3 months (rabbit and rat) or 6 months (rabbit) in comparison to the respective machined control implants could be observed. Recently, with respect to the treatment of large bone defects, a porous cast titanium alloy (Ti6Al7Nb) was reported to possess good in vitro and in vivo biocompatibility after acid etching [20]. We are not aware, however, of any previous in vitro studies on the cell-biologic response of osteogenic cells in contact with cast titanium or titanium alloy without mechanical or chemical removal of the superficial layer.

To address this knowledge gap in a clinically relevant manner, we used the established optimized casting process for Ti6Al4V, an aluminum oxide blasting process currently included in the commercial medical implant production and tested the interaction of primary human osteoblasts from patients with end-stage knee osteoarthritis who received joint-replacement surgery as a cell source. Moreover, a broad set of relevant central cell-biologic outcome parameters, such as osteoblast adhesion and proliferation, markers of early and late osteogenic di fferentiation, as well as secretion of signaling molecules reflecting bone turnover, was analyzed.

For both reference and cast material, we observed relatively large variation in the cell-biologic outcome parameters. This can be attributed to the use of primary human osteoblast cultures from di fferent patients and is a common observation for non-cell lines. Nevertheless, testing primary human cells instead of using commercially available (immortalized or tumor-derived) cell-lines has been recommended in the literature [21].

Regarding cell adhesion and the proliferation of human osteoblasts on the tested specimen of spin cast or machined Ti6Al4V material, no di fference was detectable. For the interpretation of these results, it is important that the process of aluminum oxide blasting resulted in nearly identical surface roughness. That the microstructure of the surface represents an essential factor for cell adhesion, proliferation and di fferentiation is well known and has been the subject of comprehensive reviews, for example by Mitra et al. [22]. Yokose and co-worker demonstrated that the surface micro topography of titanium discs influences osteoblast-like cell proliferation and di fferentiation [23]. Moreover, Hatano et al. reported that primary rat osteoblasts showed higher proliferation, and alkaline phosphatase and osteocalcin expression cultivated on rough rather than smooth tissue culture polystyrene [24]. It is also known from the literature that roughness of the surface is important for a successful osseointegration [25]. In addition to classical micro and nano topography, the presence of pores within the surface also has a major influence on cell adhesion and di fferentiation of the desired cells [22]. Thus, further improvement of integration can be achieved, for example, by using multi scale porous titanium

surfaces rather than smooth or micro-roughened titanium [25]. According to results from Anselme and coworkers, our findings indicate a similar biocompatibility of the reference and cast material since the surface topography, generated by classical aluminum-oxide blasting, was not di fferent [26,27]. As previously reported by Lagonegro et al., we observed that human osteoblasts preferentially adhered to the peaks of micro-topography and bridged over geometric surface irregularities [23,28].

A similar observation was published by Yin et al. [29]. The authors showed that the blasting or etching of a titanium surface has a significant e ffect on osteoblast di fferentiation. Rough-blasted surfaces supported the di fferentiation process while the etching process reduced the expression of osteoblast markers like RUNX2, COL1a1, and ALP. We also tested these osteogenic di fferentiation markers on human primary osteoblasts and found no significant di fference between the reference machined material and the cast specimen. The same result was obtained for the expression of BGLAP as a late marker of osteoblast phenotype and the in vitro mineralization determined by Alizarin Red Assay. These findings are in line with the recently published results of Wölfle-Roos et al. [4]. They showed that in rats in vivo no negative e ffect with respect to bone-implant contact or pull-out-force could be attributed to the optimized manufacturing process, which was also used in the present in vitro experiments.

Some titanium surfaces in the in vitro study were further modified by ion beam implantation of Ca- or P-ions. Comparing our data with the available literature on similar surface modifications, some divergences need to be discussed. Krupa et al. presented similar results for human bone derived cells when seeded on mirror polished pure commercial Ti-surfaces. They used similar conditions for ion implantation and found no adverse biological e ffects in terms of cell viability and ALP analysis [30,31]. In addition, Nayab et al. could not detect any e ffect on MG63 cells after seeding on pure Ti-surfaces with implanted Ca ions (ion dose 1 × 10−<sup>15</sup> or 1 × <sup>10</sup>−16). Only by using higher concentrations of Ca (ion dose 1 × <sup>10</sup>−17) did they describe slightly better cell spreading, associated with delayed adhesion and enhanced expression of bone cell markers [32,33]. Several working groups could demonstrate a positive effect on cellular behavior by coating titanium surfaces with calcium or phosphor [34–37]. Besides the di fferent metallic materials, pure titanium vs. Ti6Al4V, they also used di fferent techniques for surface modification like plasma spraying or chemical methods. Therefore, the amount of biologically available ions is not comparable [33]. By using ion implantation, the ions become dispersed into a certain range of depth and a relevant part of them becomes not bioavailable [13]. Thus, besides ion density, implantation energy also influences biological impact. Moreover, the vast majority of in vitro studies are not based on sand-blasted or etched surfaces with a roughness comparable to the current clinical application. In the case of a relevant surface micro-roughness plasma, ion implantation of calcium, used in the study of Cheng et al., could also have advantages [38]. The authors found significant positive e ffects of calcium plasma immersion ion implantation on the osteoblast-like cell line MG63 with respect to adhesion, proliferation and osteogenic di fferentiation in vitro and in a rabbit in vivo model. However, besides the di fferent ion implantation technique and dosage, the use of pure machined titanium, a lower degree of surface micro-roughness and the analysis of a bone-tumor derived cell-line instead of primary human osteoblasts in this study precludes a direct comparison. The encouraging results nevertheless indicate that di fferent strategies and dosages of ion implantation should be further studied with our optimized spin cast Ti6Al4V-alloy.

Additionally, the charge of the surface modulates cellular activities. In our study, the addition of Ca- or P-ions by ion beam implantation even negatively a ffected mineral deposition in the synthesized collagen matrix, as shown by Alizarin red S staining. This is somewhat contrary to the so far known literature [36,39]. One explanation could be the fact that these groups used di fferent methods for surface modifications than we did. They used chemical approaches or, like Knabe et al., plasma spraying to cover the titanium with Ca- or Zn-P-ions [35]. It could be possible that the surface charge was not changed in an analogous manner in addition to a di fferent amount of deposition and the bioavailability of the ions when using these alternative surface modification techniques. In future experiments, a similar approach to investigating surface charge changes as described by Kulkarni

and co-workers could be used [40]. They presented data on how surface characteristics influence interaction with small sized model proteins. Namely, variations in charge densities towards the top edges of the surface seem to be a relevant factor. This effect was shown to be mainly triggered by nano topography [41]. Due the identical production process of our cast specimen and the not significantly different results of Ra and Rz without/with ion implantation, we conclude that any possible differences in the charge density in the case of ion implantation could be due to the deposition itself or to influences on the nanostructure, which was not analyzed in this study. Future experiments should consider studying nano-topographic features and analysis of wettability as well as zeta potential.

In terms of cell apoptosis, we tested for the gene expression of CASP3, a key member of the apoptotic pathway in osteogenic cells undergoing differentiation [42]. Even using the same alloy for reference and cast specimens, treating them in the same medical grade production process, including aluminum oxide blasting and sterilization procedures with the same machinery, it could have been possible that different micro- or nano-topographic structures could have led to unwanted cell death of the osteoblasts. This relevant topic has been disused by several groups. Kulkarni et al. showed that the titanium alloy surface topography influences cell survival and cell death [43]. Unfortunately, the rough micro-topography of the specimen did not allow for a reliable measurement of the nano-topography that could somehow be different in the machined references and the cast specimen. Besides that, some knowledge arises that cytotoxic effects of Titanium and its alloys have to be considered [44]. Nevertheless, Ti6Al4V is the most frequently and successfully used alloy in orthopedic surgery because of its biocompatibility and certain bone-similar characteristics. Therefore, the present work focused on the transfer of the routine fabrication of this alloy to a newly developed fabrication method.

It was also reported that, besides the overexpression of CASP3, which leads to unwanted cell death, a loss of CASP3 expression results in delayed ossification and decreased bone mineral density. Comparing our reference material with that of the cast, we could conclude that the given CASP3 expression does not impair the osteogenic differentiation process and preserves the viability of osteoblasts. Also, our data indicate no respective difference in CASP3 expression between the tested specimens.

Because of the non-loaded in vitro conditions, a possible impact on inflammatory processes should be further tested by including, for example, abrasion experiments and by studying interaction with macrophages. Under unloaded in vitro conditions, the analysis of osteoblast OPG and RANKL expression on the gene and protein level did not indicate any disadvantage of the optimized casting process in terms of secretion of central regulators of osteoclast activity in the present study. These results are in line with our data from the in vivo study in which no relevant induction of inflammation or subsequent bone resorption or local osteolysis was observed after the implantation of small rods in the tibia metaphysis of rats [4]. Nevertheless, it should be kept in mind that further testing in a load-bearing large animal model still has to be performed.

The optimized casting process was successfully developed in terms of methods and practical procedure and is currently the subject of further research, aiming to create a scalable production process for market-ready implants. So far, produced tibia and femur prototypes meet given demands regarding microstructure as well as mechanical and machining properties. Approaching industrial part production, the process is now in the development stage to facilitate upscaled production lots for the fabrication of uncemented endoprostheses at competitive market prices. Besides the still-required increase in productivity, the improvement of dimensional accuracy is being addressed as a secondary challenge as methods to correct it are successfully employed regularly. Finally, in addition to comparison with standard machined Ti6Al4V, material comparative testing of spin cast Ti6Al4V with other Titanium alloys, like Ti6Al7Nb or Ti28NB35.4Zr, should be performed.
