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COMP-angiopoietin1 potentiates the effects of bone morphogenic protein-2 on ischemic necrosis of the femoral head in rats.

Zhou L, Yoon SJ, Jang KY, Moon YJ, Wagle S, Lee KB, Park BH, Kim JR - PLoS ONE (2014)

Bottom Line: Radiographs obtained six weeks after injection revealed better preservation of the architecture of the femoral head in the COMP-Ang1 + BMP-2 group compared with the BSA and BMP-2 groups.Histological findings indicated increased trabecular bone and vascularity and decreased osteoclast bone resorption activity in the COMP-Ang1 + BMP-2 group compared with those in the BSA and BMP-2 groups.These results suggest that COMP-Ang1 preserved the necrotic femoral head through the potentiation of BMP-2 signaling pathways and angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Orthopedic Surgery, Chonbuk National University Medical School, Research Institute for Endocrine Sciences and Research Institute of Clinical Medicine, Jeonju, Republic of Korea; Department of Sports Medicine, Taishan Medical University, Shandong, China.

ABSTRACT
Angiogenesis is considered essential for proper bone regeneration. The purpose of this investigation was to determine if a combined therapy of bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein angiopoietin-1 (COMP-Ang1) can potentiate the therapeutic effect of BMP-2 in a rat model of ischemic necrosis of the femoral head (INFH). INFH was surgically induced in the femoral head of rats, and the animals were divided into the following groups: 1) a sham-operated group (sham group), 2) a bovine serum albumin-injected group (BSA group), 3) a BMP-2-injected group (BMP-2 group), and 4) a COMP-Ang1 and BMP-2-injected group (COMP-Ang1 + BMP-2 group) (n = 20/group). Radiologic, histologic, and histomorphometric assessments were performed to assess femoral head morphology, vascular density, and bone resorption activity. Western blots and immunohistochemical staining were performed to evaluate production of BMP-related signaling proteins in C3H10T1/2 cells and tissues. Real-time RT-PCR was performed to investigate expression of the target integrin gene, and the effect of integrin on C3H10T1/2 cells was determined using a cell adhesion assay. Radiographs obtained six weeks after injection revealed better preservation of the architecture of the femoral head in the COMP-Ang1 + BMP-2 group compared with the BSA and BMP-2 groups. Histological findings indicated increased trabecular bone and vascularity and decreased osteoclast bone resorption activity in the COMP-Ang1 + BMP-2 group compared with those in the BSA and BMP-2 groups. The combination of COMP-Ang1 and BMP-2 increased phosphorylation of Smad1/3/5, p38, and Akt. Increased integrin α3 and β1 mRNA expression in the COMP-Ang1 + BMP-2 group promoted cell adhesion. These results suggest that COMP-Ang1 preserved the necrotic femoral head through the potentiation of BMP-2 signaling pathways and angiogenesis. Combination treatment with COMP-Ang1 and BMP-2 may be a clinically useful therapeutic application in INFH.

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Representative micro-CT scan images of infarcted femoral heads in the BSA, BMP-2, and COMP-Ang1 + BMP-2 groups.Shown are femoral heads obtained from the infarcted area eight weeks after surgery-induced avascular necrosis. Femoral heads showed significant loss of trabecular bone and deformity in the BSA group (B, F, J, N). Femoral heads in the BMP-2 group (C, G, K, O) showed a prominent bone resorption area, but maintained femoral head shape as compared with the BSA group. The trabecular network and femoral head architecture were well preserved in the COMP-Ang1 + BMP-2 group (D, H, L, P). The ROIs (solid red lines) of each group (M, N, O, P) were obtained from the cross-sectional area of the middle of the femoral head (dotted red lines). The sagittal images (I, J, K, L) were formed by the cross-sectional area of the blue lines perpendicular to the dotted red lines, and the coronal images (E, F, G, H) were formed by the cross-sectional area of yellow lines.
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pone-0110593-g001: Representative micro-CT scan images of infarcted femoral heads in the BSA, BMP-2, and COMP-Ang1 + BMP-2 groups.Shown are femoral heads obtained from the infarcted area eight weeks after surgery-induced avascular necrosis. Femoral heads showed significant loss of trabecular bone and deformity in the BSA group (B, F, J, N). Femoral heads in the BMP-2 group (C, G, K, O) showed a prominent bone resorption area, but maintained femoral head shape as compared with the BSA group. The trabecular network and femoral head architecture were well preserved in the COMP-Ang1 + BMP-2 group (D, H, L, P). The ROIs (solid red lines) of each group (M, N, O, P) were obtained from the cross-sectional area of the middle of the femoral head (dotted red lines). The sagittal images (I, J, K, L) were formed by the cross-sectional area of the blue lines perpendicular to the dotted red lines, and the coronal images (E, F, G, H) were formed by the cross-sectional area of yellow lines.

Mentions: A SKYSCAN 1076 Micro-CT unit (Skyscan, Kontich, Belgium) was used to assess bone volume within the defect site. The unit is intended for non-invasive imaging of the internal microstructures of objects with micrometer resolution and consists of a microfocus X-ray source, a precision object manipulator, and high resolution CCD detectors. The source was of the open tube type and the minimum focal spot size was <5 m. The spatial resolution of the micro-CT device can extend up to 4000 × 2600 pixels. In the present study, the X-ray source was set at 75 kV and 100 µA, with a pixel size at 8.8 µm, and 400 projections were acquired over an angular range of 180° (angular step of 0.45°). The image slices were reconstructed using cone-beam reconstruction software based on the Feldkamp algorithm (Dataviewer; Skyscan, Belgium). The area included in the CT scans extended from the upper margin of the epiphysis of the femoral head to femoral neck. A global thresholding algorithm was applied at a constant threshold for all specimens. This threshold was chosen as the intensity (gray value) that corresponded to ∼45% of the average intensity of the intact cortical bone in the specimens. Voxels with intensities higher than the threshold were considered to contain mineralized tissue. A constrained 3D Gaussian filter (filter width = 0.8, filter support = 1 voxel) was used to partially suppress image noise. All aspects of the system were operated using software (Dataviewer; Skyscan, Belgium). On the stacked reconstructed micro-CT cross-section images, manual regions of interest (ROIs) of an irregular anatomical contour were drawn on the transverse image at middle of the femoral head (Figure 1). The volume of interest (VOI) consisted of a stack of ROIs drawn over 52 cross-sections, resulting in a height of 0.45 mm. The VOI included the subchondral trabecular bone, the secondary ossification center, and the growth plate in the ischemic femoral head, excluding the non-ischemic femoral neck and intertrochanteric area distal to the ligation of the femoral neck. The following three-dimensional (3D) morphometric parameters were calculated for the VOI of the femoral head (Skyscan, Kontich, Belgium): bone volume (BV, mm3), bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm) and trabecular number (Tb.N, mm−1). BV is the 3D of the structure segmented as bone, and BV/TV is the ratio of the segmented bone volume to the total volume of the region of interest. Tb.Th is the mean thickness of the trabeculae, Tb.Sp is the mean distance between trabeculae, and Tb.N is the average number of trabeculae present per unit length. Tb.Th and Tb.Sp were assessed using direct 3D methods, and Tb.N was calculated using the formula Tb.N  =  (BV/TV)/Tb.Th.


COMP-angiopoietin1 potentiates the effects of bone morphogenic protein-2 on ischemic necrosis of the femoral head in rats.

Zhou L, Yoon SJ, Jang KY, Moon YJ, Wagle S, Lee KB, Park BH, Kim JR - PLoS ONE (2014)

Representative micro-CT scan images of infarcted femoral heads in the BSA, BMP-2, and COMP-Ang1 + BMP-2 groups.Shown are femoral heads obtained from the infarcted area eight weeks after surgery-induced avascular necrosis. Femoral heads showed significant loss of trabecular bone and deformity in the BSA group (B, F, J, N). Femoral heads in the BMP-2 group (C, G, K, O) showed a prominent bone resorption area, but maintained femoral head shape as compared with the BSA group. The trabecular network and femoral head architecture were well preserved in the COMP-Ang1 + BMP-2 group (D, H, L, P). The ROIs (solid red lines) of each group (M, N, O, P) were obtained from the cross-sectional area of the middle of the femoral head (dotted red lines). The sagittal images (I, J, K, L) were formed by the cross-sectional area of the blue lines perpendicular to the dotted red lines, and the coronal images (E, F, G, H) were formed by the cross-sectional area of yellow lines.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4201557&req=5

pone-0110593-g001: Representative micro-CT scan images of infarcted femoral heads in the BSA, BMP-2, and COMP-Ang1 + BMP-2 groups.Shown are femoral heads obtained from the infarcted area eight weeks after surgery-induced avascular necrosis. Femoral heads showed significant loss of trabecular bone and deformity in the BSA group (B, F, J, N). Femoral heads in the BMP-2 group (C, G, K, O) showed a prominent bone resorption area, but maintained femoral head shape as compared with the BSA group. The trabecular network and femoral head architecture were well preserved in the COMP-Ang1 + BMP-2 group (D, H, L, P). The ROIs (solid red lines) of each group (M, N, O, P) were obtained from the cross-sectional area of the middle of the femoral head (dotted red lines). The sagittal images (I, J, K, L) were formed by the cross-sectional area of the blue lines perpendicular to the dotted red lines, and the coronal images (E, F, G, H) were formed by the cross-sectional area of yellow lines.
Mentions: A SKYSCAN 1076 Micro-CT unit (Skyscan, Kontich, Belgium) was used to assess bone volume within the defect site. The unit is intended for non-invasive imaging of the internal microstructures of objects with micrometer resolution and consists of a microfocus X-ray source, a precision object manipulator, and high resolution CCD detectors. The source was of the open tube type and the minimum focal spot size was <5 m. The spatial resolution of the micro-CT device can extend up to 4000 × 2600 pixels. In the present study, the X-ray source was set at 75 kV and 100 µA, with a pixel size at 8.8 µm, and 400 projections were acquired over an angular range of 180° (angular step of 0.45°). The image slices were reconstructed using cone-beam reconstruction software based on the Feldkamp algorithm (Dataviewer; Skyscan, Belgium). The area included in the CT scans extended from the upper margin of the epiphysis of the femoral head to femoral neck. A global thresholding algorithm was applied at a constant threshold for all specimens. This threshold was chosen as the intensity (gray value) that corresponded to ∼45% of the average intensity of the intact cortical bone in the specimens. Voxels with intensities higher than the threshold were considered to contain mineralized tissue. A constrained 3D Gaussian filter (filter width = 0.8, filter support = 1 voxel) was used to partially suppress image noise. All aspects of the system were operated using software (Dataviewer; Skyscan, Belgium). On the stacked reconstructed micro-CT cross-section images, manual regions of interest (ROIs) of an irregular anatomical contour were drawn on the transverse image at middle of the femoral head (Figure 1). The volume of interest (VOI) consisted of a stack of ROIs drawn over 52 cross-sections, resulting in a height of 0.45 mm. The VOI included the subchondral trabecular bone, the secondary ossification center, and the growth plate in the ischemic femoral head, excluding the non-ischemic femoral neck and intertrochanteric area distal to the ligation of the femoral neck. The following three-dimensional (3D) morphometric parameters were calculated for the VOI of the femoral head (Skyscan, Kontich, Belgium): bone volume (BV, mm3), bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular separation (Tb.Sp, μm) and trabecular number (Tb.N, mm−1). BV is the 3D of the structure segmented as bone, and BV/TV is the ratio of the segmented bone volume to the total volume of the region of interest. Tb.Th is the mean thickness of the trabeculae, Tb.Sp is the mean distance between trabeculae, and Tb.N is the average number of trabeculae present per unit length. Tb.Th and Tb.Sp were assessed using direct 3D methods, and Tb.N was calculated using the formula Tb.N  =  (BV/TV)/Tb.Th.

Bottom Line: Radiographs obtained six weeks after injection revealed better preservation of the architecture of the femoral head in the COMP-Ang1 + BMP-2 group compared with the BSA and BMP-2 groups.Histological findings indicated increased trabecular bone and vascularity and decreased osteoclast bone resorption activity in the COMP-Ang1 + BMP-2 group compared with those in the BSA and BMP-2 groups.These results suggest that COMP-Ang1 preserved the necrotic femoral head through the potentiation of BMP-2 signaling pathways and angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Orthopedic Surgery, Chonbuk National University Medical School, Research Institute for Endocrine Sciences and Research Institute of Clinical Medicine, Jeonju, Republic of Korea; Department of Sports Medicine, Taishan Medical University, Shandong, China.

ABSTRACT
Angiogenesis is considered essential for proper bone regeneration. The purpose of this investigation was to determine if a combined therapy of bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein angiopoietin-1 (COMP-Ang1) can potentiate the therapeutic effect of BMP-2 in a rat model of ischemic necrosis of the femoral head (INFH). INFH was surgically induced in the femoral head of rats, and the animals were divided into the following groups: 1) a sham-operated group (sham group), 2) a bovine serum albumin-injected group (BSA group), 3) a BMP-2-injected group (BMP-2 group), and 4) a COMP-Ang1 and BMP-2-injected group (COMP-Ang1 + BMP-2 group) (n = 20/group). Radiologic, histologic, and histomorphometric assessments were performed to assess femoral head morphology, vascular density, and bone resorption activity. Western blots and immunohistochemical staining were performed to evaluate production of BMP-related signaling proteins in C3H10T1/2 cells and tissues. Real-time RT-PCR was performed to investigate expression of the target integrin gene, and the effect of integrin on C3H10T1/2 cells was determined using a cell adhesion assay. Radiographs obtained six weeks after injection revealed better preservation of the architecture of the femoral head in the COMP-Ang1 + BMP-2 group compared with the BSA and BMP-2 groups. Histological findings indicated increased trabecular bone and vascularity and decreased osteoclast bone resorption activity in the COMP-Ang1 + BMP-2 group compared with those in the BSA and BMP-2 groups. The combination of COMP-Ang1 and BMP-2 increased phosphorylation of Smad1/3/5, p38, and Akt. Increased integrin α3 and β1 mRNA expression in the COMP-Ang1 + BMP-2 group promoted cell adhesion. These results suggest that COMP-Ang1 preserved the necrotic femoral head through the potentiation of BMP-2 signaling pathways and angiogenesis. Combination treatment with COMP-Ang1 and BMP-2 may be a clinically useful therapeutic application in INFH.

Show MeSH
Related in: MedlinePlus