Osseointegrative effect of rhBMP-2 covalently bound on a titan-plasma-spray-surface after modification with chromosulfuric acid in a large animal bone gap-healing model with the Göttingen minipig

Bone morphogenetic proteins play an important role in the proliferation, differentiation, and induction of mesenchymal stem cells as well as in the embryogenesis [1]. BMP-2, currently one of the best-studied bone morphogenetic proteins, is applied increasingly in orthopedic surgery [2]. The working group of Jennissen developed a method for the surface modification which allows a subsequent biocoating by proteins, e.g., BMP-2 [3]. The basis of this principle underlies the production of an ultra-hydrophilic surface on titanium implants with chromosulfuric acid, which leads to a so-called inverse lotus effect, which causes a reduction of the contact angle to < 10° [4]. The procedure has already been published several times in detail and is patented [3, 5‐8]. By the addition of anchor molecules, BMP-2 can be bound and retains its biological activity [6, 9]. Thus, the prerequisite for adequate protein binding is the prior treatment with chromosulfuric acid. Previous studies showed that the treatment with chromosulfuric acid alone has an osseointegrative potential [10]. BMPs as differentiation factors are suspected to promote malignant degeneration when used in humans [11].

Gap models have been used since 1980 and earlier in orthopedic research, allowing the investigation of bone repair under special conditions with different questions [10, 12‐15].

The animal of our choice, the Göttingen minipig, has been proven to be a reliable animal for examining implants, especially because of the similar characteristics of the mesenchymal stem cells [16], as well as because of a comparable lamellar bone structure [17].

Histomorphometry, as well as the polychrome labeling, finds application in the evaluation of bone remodeling processes [10]. For fluorescence microscopy, in the sense of intravital staining, combinations of different fluorochromes lead to a better resolution of ossification fronts by synergistic effects [18], and this could be shown, e.g., for tetracycline and xylenol orange [19]. For some substances, no spectral analysis is needed, which would require an immense effort for the evaluation [20, 21].

The gold standard in orthopedic research is the histomorphometry, particularly for the assessment of osseointegration, which allows a quantitative assessment on the basis of an established standardized nomenclature since the 1980s [22‐24].

The aim of the study was to investigate the osseointegrative force of different titanium surfaces at different postoperative times in respect to a covalently bound layer made of BMP-2 additionally. The application in an animal bone gap-healing model should reveal the potential for osseoinduction and osseoconduction.

The animal study was approved by the ethical committee of the Regierungspräsidium Karlsruhe, Abteilung 3 - Landwirtschaft, Ländlicher Raum, Veterinär - und Lebensmittelwesen, Karlsruhe, Germany, under the file no. 35-9185.81/G-27/05.

The aim of the study was to evaluate the osseointegrative force of rhBMP-2 in a large animal.

We have seen the main effect in the formation of osteoid and bone at the surface of the implant less inside the gap. And the effect of the BMP-2 protein seems to be pronounced more for the growth of osteoid as a precursor of bone than at mature bone. Seen from a temporal perspective, one can assume that the appearance of osteoid is close to the recruitment of preosteoblasts. In the present study, we found an indication of new osteoid formation at the beginning after 4 weeks which we interpret as osteoinduction. This can be correlated to the explanations of Albrektsson and Johannson [35]. They point out that the osteoinduction may start immediately after injury (in the present study the implantation) with a high activity over the first weeks as the recruitment of preosteoblasts may not be evident until some weeks later. This may explain also our results that we did not see any further effects in the gap in terms of osteoinduction later on.

Several chemotactic properties have been demonstrated for BMP-2 which is discussed as responsible for attracting corresponding effector cells [36‐39]. Presumably, this property contributes to the influence of osteoconduction after long-term fixation of the BMP-2 to surfaces [5]. Since the significant differences were found mainly in the immature bone-like osteoid volume and osteoid ongrowth in the present study, it can be assumed that the effects of BMP-2 have not yet been completely assessed as the observation time was limited to 12 weeks. Further investigations should be performed to show effects at a later time.

The used BMP-2 coating was covalently bound to the titanium TPS surface by prior treatment with chromosulfuric acid and binding via anchoring molecules and maintains a contact angle of 0–8° for 50 days [25]. The biological activity is reported as still evident [25] shown in cell culture by detection of alkaline phosphatase as an indicator of bone induction by BMP-2. Thus, the results of the present study can close the gap between in vitro and in vivo assessment together with preliminary studies with the same bioactive layer [40]. Sumner and coworkers made a study to analyze the influence of different dosage of BMP-2 on top of a HA/TCP. They found a 2–3.5-fold increase of bone formation in the gap which was 3 mm in height [13]. They compared a previously reported group with HA/TCP to recent groups with locally delivered rhBMP-2 in the left humeri and implants with solely buffer in the right humeri. Effects on the contralateral site were observed with an enhanced bone ingrowth. So, Sumner and colleagues hypothesized that not the BMP-2 but rather factors released by the regeneration process are responsible for this reported contralateral effect. Thus, a systemic effect could be discussed using BMP-2 as implant coating. In the present study, the control groups were separate pigs with only BMP-2 or CSA modification or none (TPS). Thus, an effect on control implants which were set in the same animal was excluded by the study design in the present trial.

As previously described, the implanted specimens were subjected to a treatment with chromosulfuric acid at high temperatures prior to biocoating with BMP-2 via anchor molecules, a process known as “surface enhancement” [41]. It has already been shown that this has a positive effect on osseoinduction as well as on osseoconduction [10, 40]. This effect, however, appears to be higher after addition of BMP-2. But, the present results show markable differences between BMP-2 covalent and CSA, which are all not significant post hoc. Considering the results of the paired comparison of CSA and TPS in particular as described by Seidling and coworkers [10], significant differences were seen for the variables osteoid ongrowth and (12 weeks) osteoid volume (4 weeks). Thus, the effect does not appear to be due solely to the surface modification.

The used gap model allows the differentiation between the osteoinductive and the osteoconductive properties of a layer on an implant in the bone [10]. Gap models are known as models for healing and turnover research in bone [14, 42, 43]. It is also known as a model to examine some sealing effects between the implant and bone [12] when a gap results between the surface of an implant and the bone stock which should be filled by bone. A sealing effect can also be an important feature of a BMP-2 coating in primary treatment with artificial joints or even with revision surgery when a loss of the bone stock came up and gaps between implant and bone are likely [10, 44, 45]. At the other hand, a gap could be seen as a carrier system like a potential reservoir between implant and bone for BMP enhancing osteogenesis, e.g., for the treatment of nonunions after bone fractures by local delivery of the protein [46, 47]. We additionally observed a significant difference for the implant site (hip vs. knee) and the variable bone volume in the three-way ANOVA. To what extent, there is an interaction between the site of implantation (knee or hip) and the surface could only be observed. Since it was not content of the present study, further observations are needed to clarify this observation.

Proteins need appropriate carrier systems when bound at the surface of an implant to keep their biological activity [3, 7, 41, 45, 48]. Several bonding techniques are described [49]. The specific technique for bonding the BMP-2 at the TPS surface of the used implants is described by Jennissen and his group [3, 41] and was performed in the present study.

Beneath the choice of the appropriate time point the gap model may have disadvantages for the analysis of the osteoinduction properties of the BMP-2 coating of implants. The BMPs were detected when the bone was implanted into the tissue like the musculus rectus abdominis or quadriceps or in bone [50]. Looking at the situation in the gap model where alloplastic material is used at the implant site blood supply has to be missed in consequence.

A perfect animal for all types of bone research may not exist as there is a high variation in, e.g., bone turnover [51]. Especially in small animals, the bone turnover seems to be much higher than in humans or large animals [51]. As previously described, the lamellar bone structure of Göttingen minipigs is nearly similar to humans, as well as the remodeling sequences [17, 52]. Furthermore, the bone marrow and marrow stem cells of Göttingen minipigs have similar characteristics to humans, especially in proliferation and morphology [16]. Not least, there is a high experience with this type of animals in orthopedic research, and they are cost-effective and easy to handle [53]. Nevertheless, none of the common study animals like dogs, pigs, or rats is fully comparable with humans [52].

The analysis of the intravital staining revealed bone turnover over the whole observation time. None of the used scores was able to distinguish between the BMP-2 and the TPS group clearly as the values laid close together combined with wide standard deviations for both groups. The wide variances may also be caused by the differing results of the examinations of the three observers (data not shown) reflecting the individual character of the evaluation. However, the activity of bone turnover was not constant in the scores over the observation time. The “general score” and the “amount score” seemed to diverge at the end of the observation time, whereas the “intensity score” shows an undulating behavior.

However, the semi-quantitative nature of the assessment must be considered. In particular, the used scores are highly dependent on the investigator. Furthermore, the light-emitting behavior of the substance in the mineralization front is quite different and depends above all on the processing and handling of the preparations [20]. Pautke et al. [20] observed no bleaching effect for the substances xylenol orange, calcein green, and rolitetracycline during storage in the dark and room temperature over a period of 6 months. Only the substance calcein blue showed a disappearance of the fluorochrome effect after 6 months [20]. Due to the experimental setup, the temporal distance between implantation and evaluation was more than 6 months; so, a bleaching cannot be safely excluded. Comparing both techniques, the histomorphometry and the intravital staining, we guess that the histomorphometry is more effective than the intravital staining in the assessment of osseointegration of implants, not least because of the low intra- and interobserver reliability.

In general, some drawbacks of BMP-2 have to be considered when used. According to the basic characteristic of the protein, ectopic bone formation may be possible after implantation in some case reports we have found in literature [54‐57]. However, the working group of the present study was able to exclude a side effect in this case in terms of new bone formation at another site of the treated animals [33].

A review, published by Carragee et al. [11], brought BMP-2 into disrepute in the application to humans. Carragee et al. described a clear study design bias with an actually 10–50-fold increased complication rate compared to the previously published data [11]. There was a 40% increased risk of adverse events compared to the conventional autologous bone graft, as well as an increased risk of malignant disease in the absence of benefit of the BMP-2 [11, 58, 59]. Since rhBMP-2 is a derivative of a differentiation factor in the mesenchymal development, malignant degeneration is not unlikely [54, 60]. A participation in tumor angiogenesis, as well as an inhibition of tumor cell apoptosis, could also be demonstrated [39].

Thus, the harmlessness of BMP-2 should be shown in further studies as this question was not the rationale of the present study dealing with the osseointegration.

Covalently bound BMP-2, applied to a titanium sample body after prior surface modification with chromosulfuric acid, had positive effects on the osseointegration in the large animal model during the observation period of 12 weeks. The osseoconduction, expressed by the variables osteoid ongrowth and bone ongrowth, was mainly influenced. Due to the lack of significant differences in the pairwise group comparison between BMP-2 and CSA, further studies should show a benefit of the BMP-2 against the sole surface modification even more clearly. The results showed that the effect is probably not explainable by the treatment with chromosulfuric acid solely rather by an additional application of BMP-2.