Molecular and structural patterns of guided bone regeneration (GBR). Experimental studies on the role of GBR membrane and bone substitute materials

The mechanisms of guided bone regeneration (GBR) and bone healing with calcium phosphate (CaP) bone substitutes are not fully understood. The major aim of this thesis was to determine the relationship between the bone formation in bone defects and the cellular distribution and activities in association with CaP materials and/or with GBR membrane. The objectives were, firstly, to examine if the different CaP substitutes induce different cellular and molecular activities, and, secondly, to investigate the mechanisms of GBR with focus on the role of the barrier membrane in the bone healing process. A series of studies were performed in a rat trabecular bone defect model using a set of molecular (e.g. qPCR) and morphological (e.g. histology & histomorphometry) techniques. Deproteinized bovine bone (DBB) and octa-CaP (TetraB) granules promoted bone regeneration and restitution of the defect. DBB was osteoconductive and elicited low resorption activity. TetraB induced early osteogenic and osteoclastic activities, resulting in greater bone formation than DBB. Strontium (Sr) doping of the CaP granules reduced the expression of osteoclastic resorption genes in comparison to hydroxyapatite (HA). Applying a collagen-based membrane on the defect promoted higher bone formation at all time periods. This was in parallel with upregulation of genes denoting cell recruitment and coupled bone formation and resorption (i.e. remodeling). The membrane was found to accumulate cells that expressed and released different pro-osteogenic growth factors (e.g. BMP-2). When the defect was simultaneously treated with the membrane and bone substitutes (DBB, HA, SrHA), more bone and an inhibitory effect of Sr on osteoclasts was demonstrated in the SrHA treated defect. In conclusion, different calcium phosphate bone substitutes induce specific molecular cascades involved in the different processes of bone healing, including early inflammation, bone formation and remodeling. This promotes bone regeneration and defect restitution in comparison with the sham defect. Strontium incorporation in a synthetic CaP substitute reduces the osteoclastic resorptive activities, and promotes bone formation. Furthermore, the present results provide cellular and molecular evidence in vivo suggesting a novel role for the membrane during GBR, by acting as a bioactive compartment rather than as a passive barrier. The results provide new opportunities for the design of a new generation of materials to enhance bone regeneration

Strontium-Doped Calcium Phosphate and Hydroxyapatite Granules Promote Different Inflammatory and Bone Remodelling Responses in Normal and Ovariectomised Rats

The healing of bone defects may be hindered by systemic conditions such as osteoporosis. Calcium phosphates, with or without ion substitutions, may provide advantages for bone augmentation. However, the mechanism of bone formation with these materials is unclear. The aim of this study was to evaluate the healing process in bone defects implanted with hydroxyapatite (HA) or strontium-doped calcium phosphate (SCP) granules, in non-ovariectomised (non-OVX) and ovariectomised (OVX) rats. After 0 (baseline), six and 28d, bone samples were harvested for gene expression analysis, histology and histomorphometry. Tumour necrosis factor-alpha (TNF-alpha), at six days, was higher in the HA, in non-OVX and OVX, whereas interleukin-6 (IL-6), at six and 28d, was higher in SCP, but only in non-OVX. Both materials produced a similar expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). Higher expression of osteoclastic markers, calcitonin receptor (CR) and cathepsin K (CatK), were detected in the HA group, irrespective of non-OVX or OVX. The overall bone formation was comparable between HA and SCP, but with topological differences. The bone area was higher in the defect centre of the HA group, mainly in the OVX, and in the defect periphery of the SCP group, in both non-OVX and OVX. It is concluded that HA and SCP granules result in comparable bone formation in trabecular bone defects. As judged by gene expression and histological analyses, the two materials induced different inflammatory and bone remodelling responses. The modulatory effects are associated with differences in the spatial distribution of the newly formed bone

Guided bone regeneration using resorbable membrane and different bone substitutes : Early histological and molecular events

Bone insufficiency remains a major challenge for bone-anchored implants. The combination of guided bone regeneration (GBR) and bone augmentation is an established procedure to restore the bone. However, a proper understanding of the interactions between the bone substitute and GBR membrane materials and the bone-healing environment is lacking. This study aimed to investigate the early events of bone healing and the cellular activities in response to a combination of GBR membrane and different calcium phosphate (CaP) materials. Defects were created in the trabecular region of rat femurs, and filled with deproteinized bovine bone (DBB), hydroxyapatite (HA) or strontium-doped HA (SrHA) or left empty (sham). All the defects were covered with an extracellular matrix membrane. Defects were harvested after 12 h, 3 d and 6 d for histology/histomorphometry, immunohistochemistry and gene expression analyses. Histology revealed new bone, at 6 d, in all the defects. Larger amount of bone was observed in the SrHA-filled defect. This was in parallel with the reduced expression of osteoclastic genes (CR and CatK) and the osteoblast-osteoclast coupling gene (RANKL) in the SrHA defects. Immunohistochemistry indicated fewer osteoclasts in the SrHA defects. The observations of CD68 and periostin-expressing cells in the membrane per se indicated that the membrane may contribute to the healing process in the defect. It is concluded that the bone-promoting effects of Sr in vivo are mediated by a reduction in catabolic and osteoblast-osteoclast coupling processes. The combination of a bioactive membrane and CaP bone substitute material doped with Sr may produce early synergistic effects during GBR. Statement of significance The study provides novel molecular, cellular and structural evidence on the promotion of early bone regeneration in response to synthetic strontium-containing hydroxyapatite (SrHA) substitute, in combination with a resorbable, guided bone regeneration (GBR) membrane. The prevailing view, based mainly upon in vitro data, is that the beneficial effects of Sr are exerted by the stimulation of bone-forming cells (osteoblasts) and the inhibition of bone-resorbing cells (osteoclasts). In contrast, the present study demonstrates that the local effect of Sr in vivo is predominantly via the inhibition of osteoclast number and activity and the reduction of osteoblast-osteoclast coupling. This experimental data will form the basis for clinical studies, using this material as an interesting bone substitute for guided bone regeneration

Strontium-Doped Calcium Phosphate and Hydroxyapatite Granules Promote Different Inflammatory and Bone Remodelling Responses in Normal and Ovariectomised Rats

The healing of bone defects may be hindered by systemic conditions such as osteoporosis. Calcium phosphates, with or without ion substitutions, may provide advantages for bone augmentation. However, the mechanism of bone formation with these materials is unclear. The aim of this study was to evaluate the healing process in bone defects implanted with hydroxyapatite (HA) or strontium-doped calcium phosphate (SCP) granules, in non-ovariectomised (non-OVX) and ovariectomised (OVX) rats. After 0 (baseline), six and 28d, bone samples were harvested for gene expression analysis, histology and histomorphometry. Tumour necrosis factor-alpha (TNF-alpha), at six days, was higher in the HA, in non-OVX and OVX, whereas interleukin-6 (IL-6), at six and 28d, was higher in SCP, but only in non-OVX. Both materials produced a similar expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). Higher expression of osteoclastic markers, calcitonin receptor (CR) and cathepsin K (CatK), were detected in the HA group, irrespective of non-OVX or OVX. The overall bone formation was comparable between HA and SCP, but with topological differences. The bone area was higher in the defect centre of the HA group, mainly in the OVX, and in the defect periphery of the SCP group, in both non-OVX and OVX. It is concluded that HA and SCP granules result in comparable bone formation in trabecular bone defects. As judged by gene expression and histological analyses, the two materials induced different inflammatory and bone remodelling responses. The modulatory effects are associated with differences in the spatial distribution of the newly formed bone

Guided bone regeneration using resorbable membrane and different bone substitutes : Early histological and molecular events

Bone insufficiency remains a major challenge for bone-anchored implants. The combination of guided bone regeneration (GBR) and bone augmentation is an established procedure to restore the bone. However, a proper understanding of the interactions between the bone substitute and GBR membrane materials and the bone-healing environment is lacking. This study aimed to investigate the early events of bone healing and the cellular activities in response to a combination of GBR membrane and different calcium phosphate (CaP) materials. Defects were created in the trabecular region of rat femurs, and filled with deproteinized bovine bone (DBB), hydroxyapatite (HA) or strontium-doped HA (SrHA) or left empty (sham). All the defects were covered with an extracellular matrix membrane. Defects were harvested after 12 h, 3 d and 6 d for histology/histomorphometry, immunohistochemistry and gene expression analyses. Histology revealed new bone, at 6 d, in all the defects. Larger amount of bone was observed in the SrHA-filled defect. This was in parallel with the reduced expression of osteoclastic genes (CR and CatK) and the osteoblast-osteoclast coupling gene (RANKL) in the SrHA defects. Immunohistochemistry indicated fewer osteoclasts in the SrHA defects. The observations of CD68 and periostin-expressing cells in the membrane per se indicated that the membrane may contribute to the healing process in the defect. It is concluded that the bone-promoting effects of Sr in vivo are mediated by a reduction in catabolic and osteoblast-osteoclast coupling processes. The combination of a bioactive membrane and CaP bone substitute material doped with Sr may produce early synergistic effects during GBR. Statement of significance The study provides novel molecular, cellular and structural evidence on the promotion of early bone regeneration in response to synthetic strontium-containing hydroxyapatite (SrHA) substitute, in combination with a resorbable, guided bone regeneration (GBR) membrane. The prevailing view, based mainly upon in vitro data, is that the beneficial effects of Sr are exerted by the stimulation of bone-forming cells (osteoblasts) and the inhibition of bone-resorbing cells (osteoclasts). In contrast, the present study demonstrates that the local effect of Sr in vivo is predominantly via the inhibition of osteoclast number and activity and the reduction of osteoblast-osteoclast coupling. This experimental data will form the basis for clinical studies, using this material as an interesting bone substitute for guided bone regeneration

Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after 28d of healing.

<p>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).</p

Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after 28d of healing.

<p>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).</p

Gene expression analysis of inflammatory and apoptosis markers.

<p>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.</p

Light micrographs of non-decalcified toluidine blue-stained ground sections of femur defects after six days of healing.

<p>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). </p

Gene expression analysis of bone formation, bone resorption and angiogenesis markers.

<p>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.</p