Morphology and Mineral Characteristics in the Fracture Callus of Rats
1
Liu, Y; 2Mehta, M; 2Strube, P; 1Manjubala, I; 1Fratzl, P; +2Duda, G N
Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam, Germany
+2Julius Wolff Institut and Center for Musculoskeletal Surgery, Charité - Universitätsmedizin Berlin, Berlin, Germany
[email protected]
1
INTRODUCTION:
Endochondral ossification in fracture healing is a complex process
where multiple tissue types are formed, remodeled and removed to reach
complete regeneration. Not much is known about the detailed material
properties of the callus and the tissue characteristics, such as the
distribution of mineral particle size and orientation. Since not only the
macroscopic but also the nanoscopic structure of the callus is critical for
the final mechanical competence and the healing outcome, rat callus
tissues were studied using X-ray micro-tomography (μCT) combined
with scanning small-angle X-ray scattering (sSAXS) and environmental
scanning electron microscopy (ESEM). The aim of this study was to
relate the nano-structure of mineralized rat callus to its macroscopic
structure and morphology. The hypothesis of this study was that callus
structure is by no means homogeneous throughout healing bone tissue. It
is rather very locally shaped to account for the differences in mechanical
constrains within various regions.
MATERIALS AND METHODS:
Bone/callus samples of female SD rats come from a previous study
[1]. The left femur was osteotomized, distracted by 1.5 mm and
stabilized with an external fixator. Variation of age (12 vs. 52 weeks)
and fixator stiffness (rigid vs. semirigid) resulted in 4 groups (n=8): YS:
young semi-rigid, OS: old semi-rigid, YR: young rigid and OR: old rigid.
Radiographical examinations were performed post-operatively and
weekly. Animals were sacrificed at 6 weeks when full healing was
achieved. Both femurs were harvested and imaged using a Scanco
Viva40 μCT at a voxel size of 12.5 μm. 3D images of pure mineralized
callus were then rendered by applying a threshold for mineralized tissue
of 550 mg HA/cm3. For the same animals, Safranin-Orange/von Kossa
staining was done on PMMA-embedded (un-decalcified) sections (Fig.
2a). Regions of interest for sSAXS were selected within cortex and
callus near the osteotomy, based on the 3D μCT and 2D histology
images.
In all groups, eight animals were analyzed except for sSAXS, where
multiple measurement points were set in a representative sample.
Sections of 200 µm thick were cut for sSAXS from PMMA-embedded
specimens. Measurements were carried out using a laboratory X-ray
instrument (Nanostar, Bruker AXS, Karlsruhe, Germany) with 200 μm
beam size, 600 mm sample-to-detector distance and 3600 seconds
accumulation time. Mean thickness (T parameter), degree of alignment
(ρ parameter) and predominant orientation (ψ parameter) of mineral
particles were deducted from resulting sSAXS patterns. ESEM imaging
with a back scattered electron (BSE) detector (FEI FEG-ESEM Quanta
600, FEI Company, USA) was also performed on the regions of interest
to correlate 2D mineral distribution of the sSAXS section with 3D
mineral distribution obtained from μCT.
animal. It was observed with ESEM that the degree of mineralization
was different in cortex and callus (Fig. 1a and 2b) indicating different
qualities of bone tissue that have formed with time. sSAXS showed that
minerals in cortex had generally larger degree of alignment (higher ρ
values) than those in callus and their alignment followed the long bone
axis (Fig. 1c and 2c) as also seen in sheep model [2]. Within callus at the
osteotomy gap, the mineral particles had orientations perpendicular to
long axis (Fig. 1c). Whist in woven callus, predominant orientation of
the minerals agreed with the local structure of trabecular (Fig. 2c).
Apart from the pre-existing cortex (primary cortex), as shown by μCT
in all the four animals, the rat callus developed a shell-like structure
(seen in Fig. 1a, 1b, 2a and 2b) covering the osteotomy gap. sSAXS data
revealed that mineral particles in the shell (secondary cortex) were
typically thicker (higher T values, Fig. 1c) but less oriented (lower ρ
values, Fig. 1c) than in the pre-existing cortex. They also showed
reduced mineral content in µCT (Fig. 1a and 1b) and ESEM (Fig. 1a and
2b). This indicates a woven bone characteristic of the shell compared to
the cortex.
Fig. 2: (a) Safranin – Orange / von Kossa section of the OR callus at 6
weeks. Shell formation around the osteotomy gap. Scale bar = 5 mm. (b)
sSAXS ROIs on ESEM-BSE overview. Pixel brightness indicates
qualitatively the local degree of mineralization at the sample surface. (c)
Distribution of mineral particle characteristics in ROIs from b. Mean
mineral thickness (T parameter) is illustrated with color contour.
Predominant mineral orientation (ψ parameter) and degree of mineral
alignment (ρ parameter) are illustrated with bars. Spacing of data points
= 200 μm. Region 1 to 5 as marked in b.
In the OR animal, pronounced endosteal ossification was observed by
ESEM and evaluated by sSAXS differentiating endoestal, cortical,
periosteal (Fig. 2b and 2c). We found that:
z
Endosteal (region 1) was significantly different in T (p=0.001)
and ρ (p=0.001) than the intracortical callus (region 5).
z
Endosteal (region 1) was significantly different in T (p<0.001)
and ρ (p<0.001) than the cortical bone (region 4).
z
Endosteal (region 1) was significantly different only in ρ
(p=0.001) in comparison to fracture gap callus (region 3)
indicating that interfragmentary shear and movement locally
affects alignment but not size of the callus minerals.
Fig. 1: (a) 3D μCT image of the YR callus registered with 2D ESEMBSE image (inset picture). (b) 3D μCT image of the same YR callus
registered with 2D histology image (inset picture). (c) sSAXS results of T
(contour plot) and ρ (bar plot) in the ROI (shown in b) reveal a callus
shell structure with higher thickness and lower degree of alignment of
mineral particles. Direction of the bars represents predominant mineral
orientation (ψ parameter), and length of the bars represents degree of
mineral alignment (ρ parameter, 0 means randomly oriented while 1
means perfectly aligned). Spacing of data points = 300 μm.
DISCUSSION:
Correlations between macroscopic morphological characteristics of
callus with nanoscopic structural properties of its minerals were found
by combining μCT, sSAXS and ESEM. Even though histological
sections and µCT suggested a rather homogeneous mineralization of the
callus tissue, the sSAXS analyses unveiled a quite distinct structuring for
the shell compared to the pre-existing cortex. It appears that there is
location-specific adaption of callus minerals presumably in response to
the location-specific healing need.
RESULTS:
The registered μCT/ESEM image and μCT/histology image of the YR
animal are shown in Fig. 1a and 1b respectively. Fig. 2a and 2b are
comparative illustrations of histology and ESEM images of the OR
REFERENCES:
[1] Mehta M et al. (2009) ORS 55th Annual Meeting
[2] Liu Y et al. (2008) Calc Tissue Int 82(Suppl. 1): S69
Poster No. 564 • 56th Annual Meeting of the Orthopaedic Research Society