[Novel software-based and validated evaluation method for objective quantification of bone regeneration in experimental bone defects].
OBJECTIVE
The quantification of newly formed bone in experimental defect models is a problem in various experimental set-ups. Several methods have been described to evaluate and quantify the regeneration of newly formed bone in various animal models. Most methods only describe the amount of regenerated tissue on a semi-quantitative level, the results significantly depend on the subjective rating of the observer and such evaluation methods have not been validated in terms of objectivity and reliability. The aim of the present study was to introduce a novel evaluation method for the accurate quantification of bone regeneration on digital X-ray images using a freely available digital image software analysis programme (GIMP, GNU General Public Licence).


METHODS
The method introduced here contains 5 steps: standardisation of size and colour, determination of range of interest (ROI), defining different qualities of mineralisation, pixel analysis with histogram function, similar to the Hondsfield index, and quantification. In order to evaluate the objectivity and reliability, the quantification method was compared to semi-quantitative scores described by Mosheiff and Werntz for inter- and intraobserver variability. Six observers were asked to determine bone regeneration in 16 X-ray images of 2 different animal models. In order to describe intraobserver variability, the evaluation was repeated after a period of 4 weeks. Statistical analysis including determination of intra- and interobserver variability (Bland-Altman coefficient of reproduction) was performed using SAS software.


RESULTS
For both experimental set-ups analysed in this project (rabbit and sheep bone defects), the objectivity was significantly higher in the GIMP-based evaluation compared to the evaluation according to Mosheiff and Werntz using the Bland-Altman coefficient (rabbit: GIMP: 0.095, Mosheiff: 0.272, Werntz: 0.283; sheep: GIMP: 0.098, Mosheiff: 0.658, Werntz: 0.668). Analogous results were obtained for reliability (rabbit: GIMP: 0.086, Mosheiff: 0.221, Werntz: 0.385; sheep: GIMP: 0.102, Mosheiff: 0.339, Werntz: 0.623).


CONCLUSIONS
This quantification method introduced here has proved to be a reliable and "easy-to-use" tool in order to perform objective quantification of bone regeneration in 2 different experimental set-ups. It offers a more detailed and quantitative way for precise determination of regenerated tissue and is characterised by higher objectivity and reliability compared to other semi-quantitative evaluation methods. The objectivity seems to be independent of the animal model to which the method is applied.
Schönberger
T
T
Department für Orthopädie und Traumatologie, Universitätsklinikum Freiburg, Freiburg.
Kasten
P
P
Fechner
K
K
Südkamp
N P
NP
Pearce
S
S
Niemeyer
P
P
D000818:Animals; D001860:Bone Plates; D001861:Bone Regeneration / Q000502:physiology*; D004195:Disease Models, Animal; D016267:External Fixators; D015588:Observer Variation; D011817:Rabbits; D011856:Radiographic Image Enhancement / Q000379:methods*; D011857:Radiographic Image Interpretation, Computer-Assisted / Q000379:methods; D011884:Radius / Q000000981:diagnostic imaging / Q000502:physiology; D012984:Software; D012986:Software Validation; D013977:Tibia / Q000000981:diagnostic imaging / Q000502:physiology; D023822:Tissue Engineering / Q000379:methods