Original article
Scintigraphic evaluation of rhBMP-2-biocoated implants reveals no ectopic bone formation

https://doi.org/10.1016/j.biopha.2010.10.008Get rights and content

Abstract

The main objectives of the study described below were of two-fold nature: (1) to examine if rhBMP-2-biocoated implants in a pig model could lead to ectopic bone formation and (2) if quantitative and/or qualitative differences could be found between adhesively and covalently bonded BMP II using the scintigraphic method. In order to examine these central questions, 26 Göttingen minipigs were allocated to three groups with a control group (n = 7) and two study groups (n = 9 each) receiving one of three implant types: (a) chromosulfuric acid treated titanium surface as control, (b) non-covalently bonded BMP-2, and (c) covalently bonded and immobilized rhBMP-2. Each animal received four barbell-shaped implants, one in the proximal and distal metaphysis of each femur. The scintigraphic analyses were conducted after four, eight, and 12 weeks postoperatively. The visual (qualitative) analysis failed to show ectopic bone formation in any of the three groups. The statistical analysis of the relative values for bone formation yielded no significant differences between the groups, although the limitation in the applied methods do not enable one to draw conclusions regarding the histomophometric results.

Introduction

Within the last decade, a whole slew of osteoinductive biological materials have been isolated and adapted for reconstructing bone defects, some of which may play a more important role in the future [1].

One of these materials, BMP, has almost exclusively been applied to its carrier substance in its soluble form, which leads to the absorption of this protein to the surface of the carrier substance [2].

A series of experiments have shown that this type of BMP application leads to increased bone formation [2], [3], [4], [5].

Jennissen was able to synthesize and immobilize large amounts of rhBMP-2 on the surface of implants [6]. Furthermore, they found that this bonded rhBMP-2 maintained its biological activity. Additionally, an animal model failed to show qualitative or quantitative differences in the ectopic bone formation of the immobilized or soluble BMP. Although not statistically significant, the immobilized BMP tended to show greater contact surfaces between the bone and the implants [7]. These results were later confirmed in an in vivo animal experiment carried out by the same working group [8].

Sumner et al. were able to demonstrate that rhBMP-2 may not only have a topic effect near the implant, but also a systemic effect. Using a gap model in dogs, they could demonstrate that the contralateral control implant showed greater activity than expected [5]. However, the analysed value of activity was based on results from a previous, although identical, animal model.

Considering the sum of these results, it seems possible that BMP-2 could have a systemic effect manifestating as a kind of ectopic bone formation. Study groups were able to demonstrate the osteoinductive capability of rhBMP II for both intramuscular as well as hypodermic osteoinduction in in vivo experiments using type 1 collagen as a carrier [9].

This subject gains in importance by the actually published literature about heterotopic ossification after the use of commercially available recombinant human BMP proteins in the treatment of nonunions and bone loss in complex fractures [10].

As there are no further systemic effects described or found in the current literature, we decided to use the nuclear medicine method of bone scintigraphy as a sensitive technique, which could give the overview of ectopic bone formation in the whole body.

Varady et al. [11] stated that scintigraphy is a viable means of visually demonstrating rhBMP II induced ectopic bone formation. Furthermore, scintigraphy of the bones has been assessed to be a diagnostic tool frequently used for differentiating bone-specific changes in the body from changes in form of inflammatory processes to metastases [12] by means of injecting radioactively marked bisphosphonates, which have a strong affinity to osteoblasts.

Within the framework of the project, the so-called Goettingen minipigs received covalently or adhesively bonded BMP-2 coated implants. The hypothesis was that this difference in bonding could potentially show different systemic effects. Using scintigraphy as a method to demonstrate ectopic effects of BMP II within the entire body, this study sought out to examine:

  • if BMP-2 coated implants lead to ectopic bone growth;

  • if a qualitative and quantitative difference exists between the non-covalently and covalently bonded BMP II.

Section snippets

Materials and methods

In order to perform animal experiments, state approval was required and granted (file number 35-9185.81/G-27/05).

In this project based on implants coated with biological substances, 36 Goettingen minipigs received a total of four barbell-shaped implants each, one in the distal and one in the proximal metaphysis of each femur.

Four different implant types were examined. One control group received a TPS-surface, one further control group received implants treated with chromosulfuric acid, which

Statistics

The statistical analysis was carried out using the SAS 8.0 (SAS, Chicago, Ill.) program in a GLM model providing a repeat measure design, as from one animal more than one data set was taken. Two analyses were performed: one regarding the implant type, the post operative time, and the localization of the implants as independent variables and one with the addition of the animal as additional variable.

Results

Directly following the scintigraphy, experts in the field of nuclear medicine examined and visually evaluated the images (Fig. 4).

For the most part symmetrical images with physiological levels of activity were found.

However, the femora themselves showed increased levels of activity both in the proximal and distal segments of implantation, as are to be expected due to increased osteointegration as a result of the postoperative healing process (Fig. 5).

A significant difference between the two

Discussion

Two main issues were addressed in this study. The first – the possible existence of ectopic bone fromation through BMP-2 implants – could be answered using qualitative criteria and the assistance of qualified radiologists. The second question – namely the appropriateness of bone scans to discover possible local differences in osteoinduction between adhesive and covalently bonded BMP-2 on titanium implants – required the creation of comparable data between the assessed pigs. As a result, the raw

Conclusion

Heterotopic bone-formation was addressed in the present study to rule out this possible adverse event using BMP-2 layers on implants. The analysis needs appropriate technique, which is found in the scintigraphic determination in an animal study with minipigs. However the procedure seems not appropriate to analyse bone turn over at the implant sites but ectopic bone formation was able to be excluded in this experiment.

Conflict of interest statement

The authors have no conflicts of interest.

Acknowledgments

We thank Mr Lingner for his support with the manuscript.

We thank Mr Forsch and Mr Steil for supporting the handling of the animals.

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