Figure 1.
Schematic diagram showing the area of measurements (histomorphometry).
A software rectangular grid consisting of twelve zones covers and determines the entire area of the treated defect. C = Central region; P = Peripheral region; OCB = Original cortical bone; OTB = Original trabecular bone; NB = new bone; G = Granule.
Figure 2.
Material characterisation, in vitro degradation and ion release.
(A and B) X-ray diffraction analysis (XRD) of the hydroxyapatite (HA) and the strontium substituted calcium phosphate (SCP) granules. (C - F) Scanning electron microscopy (SEM) images of the HA (C, D) and SCP (E, F) granules. (G) In vitro degradation profile of HA and SCP granules. (H) Release of Ca, P, Mg and Sr ions from HA (top) and SCP (bottom) granules during 3, 7, 14 and 28d.
Figure 3.
Gene expression analysis of inflammatory and apoptosis markers.
The analysis was performed on the tissue harvested from defects filled with hydroxyapatite (HA) and strontium-doped calcium phosphate (SCP) in non-OVX and OVX rats after six and 28d of implantation. Expression levels of inflammatory markers TNF-α (A) and IL-6 (B) and apoptosis marker Caspase 3 (C). Statistically significant differences (p < 0.05) are indicated by the small letters: a = significant difference between baseline non-OVX and non-OVX (HA or SCP; 6 or 28d); b = significant difference between baseline OVX and OVX (HA or SCP; 6 or 28d); c = significant difference between HA and SCP (non-OVX or OVX; 6 or 28d); d = significant difference between non-OVX and OVX (HA or SCP; 6 or 28d); e = significant difference between six days and 28d (HA or SCP; non-OVX or OVX). The results are presented as the mean ± SEM.
Figure 4.
Gene expression analysis of bone formation, bone resorption and angiogenesis markers.
The analysis was performed on the tissue harvested from defects filled with hydroxyapatite (HA) and strontium-doped calcium phosphate (SCP) in non-OVX and OVX rats after six and 28d of implantation. Expression levels of Col1a1 (A), ALP (B), OC (C), OPG (D), RANKL (E), CR (F), CatK (G) and VEGFA (H) Statistically significant differences (p < 0.05) are indicated by the small letters: a = significant difference between baseline non-OVX and non-OVX (HA or SCP; 6 or 28d); b = significant difference between baseline OVX and OVX (HA or SCP; 6 or 28d); c = significant difference between HA and SCP (non-OVX or OVX; 6 or 28d); d = significant difference between non-OVX and OVX (HA or SCP; 6 or 28d); e = significant difference between six days and 28d (HA or SCP; non-OVX or OVX). The results are presented as the mean ± SEM.
Figure 5.
Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after six days of healing.
The defects were created in non-ovariectomised (non-OVX) (A-G) or ovariectomised (OVX) (H-N) rats. The defects were filled with either hydroxyapatite (HA) (A-D and H-J) or strontium calcium phosphate (SCP) (E-G and K-N) granules. The survey images for each group (A = non-OVX HA, E = non-OVX SCP, H = OVX HA, K = OVX SCP) show that the granules are generally within the defect boundaries in the trabecular area of the femoral epiphysis. During this time period, no major difference in the amount of granules was observed between the four different groups. However, a more defined shape of the HA granules is observed in contrast to the SCP, which have a homogeneous appearance with less well defined individual granules. All the defects appear to be largely occupied by the granules in contact with the original bone and bone marrow (BM) at the periphery of the defect. No evident bone formation at this magnification level is observed. The cut borders of the original cortical bone layer (OCB) and the original trabecular bone (OTB) are still clearly visible, with minimum signs of remodelling or outward bone formation. In the higher magnification images, multinucleated giant cells (indicated by black arrows) were very commonly detected at the surface of larger granules. In the HA groups, the multinucleated cells appear to be surrounding some smaller particles in the micron range (HAp) (D and I). Spindle-shaped mononuclear cells (macrophage-like cells; some are indicated by yellow arrows) are also detected in relation to granules and particles in all treatment groups (D, G and I). At high magnification, signs of bone formation are detected with osteoblast seams (indicated by black arrowheads) forming osteoid (Os) appearing directly on some HA granules (B and J) and at some distance from or in between the SCP granules (N). Numerous blood vessels (BV) are observed, especially in the SCP group (G).
Figure 6.
Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after 28d of healing.
The defects were created in non-ovariectomised (A-F) or ovariectomised (G-L) rats. The defects were filled with either hydroxyapatite (HA) (A-C and G-I) or strontium calcium phosphate (SCP) (D-F and J-L). The survey images for each group (A = non-OVX HA, D = non-OVX SCP, G = OVX HA, J = OVX SCP) show the pattern of new bone formation (NB) and the distribution of the remaining granules within the defect boundaries. An evident amount of separated granules with various sizes and shapes appears within the HA defects (A and G) and, at least at this magnification, new bone (NB) and bone marrow areas (BM) are more visible in the SCP defects (D and J). The demarcation line between the old bone (OB) and the newly formed bone (NB) at the defect borders is sometimes hardly defined (A and G), but it is well defined in some defects (D). (B, E, H and K) show that the granules are surrounded and interconnected by mature bone (MB) in the central region of the four different defects. A considerable amount of mature bone has formed in the central region of HA defects (B and H), in contrast to the central region of SCP defects (E and K). Multinucleated giant cells (some indicated by black arrows) were detected at the granule surface (H and K). (C, F, I and L) show that the granules are also surrounded and interconnected by mature bone in the peripheral region of the four different groups. The peripheral region of the HA defects are largely occupied by the remaining granules with less mature bone (C and I) and a more mature bone area in the peripheral region of SCP defects (F and L).
Figure 7.
Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after 28d of healing.
The defects were created in non-ovariectomised (A-G) or ovariectomised (H-Q) rats. The defects were filled with either hydroxyapatite (HA) (A-D and H-L) or strontium calcium phosphate (SCP) (E-G and M-Q). Mature bone (MB) is formed around and in contact with the granule surface in the four different groups (A = non-OVX HA, E = non-OVX SCP, H and I = OVX HA, M = OVX SCP). Bone formation is clearly visible, with osteoblast seams (indicated by black arrowheads) forming osteoid (Os) appearing directly on some HA granules (B), while mature bone (MB) lined with osteoblasts is observed on the SCP granules (P). A series of osteoblast seams is clearly observed enclosing some granule remnants in the SCP non-OVX defect (F). In the HA OVX defect, signs of bone remodelling are obvious, with the formation of multinucleated cells, blood vessels (BV) and osteoblast seams between the mature bone and granule surface (J). Multinucleated giant cells (indicated by black arrows) were commonly detected at the surface of HA granules (C, D, K and L) and, to a lesser extent, on the SCP granules (G and Q). Spindle-shaped mononuclear cells (macrophage-like cells) (some are indicated by yellow arrows) were also detected in all groups (G, K and Q).
Figure 8.
The graphs show the granule (left) and bone (right) area percentages (%) after six and 28d of implantation. Percentages (%) of granule and bone areas were calculated at total (A, D), central (B, E) and peripheral (C, F) levels of the defects. Each box plot shows the mean, median, standard deviation and the range of measurements (n = 8). Statistically significant differences (p < 0.05) are indicated by the small letters: a = significant difference between HA and SCP (at each time point for each animal group); b = significant difference between non-OVX and OVX (at each time point for each material group); c = significant difference between six and 28 d (for each group).