Neovascularization of osteoporotic metaphyseal bone defects: A morphometric micro-CT study.

PURPOSE: Neovascularization is essential for bone regeneration in fractures. This study aimed to investigate the microvascular morphology and distribution in the non-injured femur and the neovascularization of the metaphyseal critical size defect in a small animal model of osteoporosis. MATERIALS AND METHODS: Female rats (n=7) were ovariectomized (OVX) and received a multideficiency diet. Three months after OVX, a 5mm wedge shaped critical size defect was cut at the distal femoral metaphysis and stabilized with a T-shaped mini-plate. After six weeks, the animals were euthanized, and femora were removed and decalcified for micro-CT measurement of fracture neovascularization. RESULTS: No fracture healing was observed along the critical size defects. In the non-injured bone, micro-vessel distribution showed a specific pattern, thereby enabling a differentiation between epi-, meta- and diaphysis. Micro-CT based morphometry revealed a significant reduction of the vascular volume fraction as well as the vascular thickness (p<0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p<0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly. CONCLUSIONS: Micro-CT based vascular morphometry demonstrated differences between epi-, meta- and diaphysis in the non-injured bone as well as differences between the critical size defect and the non-injured metaphysis. As angiogenesis is a crucial prerequisite that precedes osteogenesis, our results may influence further evaluation of osteoconductive or osteogenic biomaterials in this small animal model of osteoporosis.

Bone status of adult female butyrylcholinesterase gene-deficient mice.

Butyrylcholinesterase (BChE) degrades acetylcholine in addition to acetylcholinesterase (AChE) which is involved in embryonic development of limbs. Since BChE is expressed by osteoblast-like cells we asked whether it is functional in adult bone remodeling. We addressed this issue by analyzing BChE gene-deficient mice (BChE-KO). Bones were extracted from 16-week old female BChE-KO and corresponding wild type mice (WT). Femoral bones were used for biomechanical testing and µCT evaluation of cancellous and cortical bone. Also vertebrae Th12 and L1 were investigated with µCT while L3 was used for tartrate-resistant acidic phosphatase (TRAP) histomorphometry and Th10 for gene expression analysis by means of real-time RT-PCR. BChE-KO did not reveal significant differences in biomechanical bone strength and bone mineral density determined by µCT. Microarchitecture of cancellous and cortical bone showed an increase in µCT parameters like trabecular thickness, trabecular separation, and relative cortical bone area of femoral BChE-KO bone compared to WT. In vertebrae no changes of microstructure and mRNA expression were detected. However, osteoclast histomorphometry with TRAP stained sections demonstrated a significant increase in relative osteoclast number. In conclusion, in adult murine bone the role of BChE is limited to bone specific changes in microarchitecture and to an increase in relative number of bone resorbing osteoclasts whereas the main collagen resorbing enzyme Cathepsin-K (CtsK) was stably expressed. Besides, AChE might be able to compensate the lack of BChE. Thus, further analyses using bone tissue specific AChE BChE cre-lox double knockout mice would be helpful.

The development and validation of micro-CT of large deep frozen specimens.

Repetitive freeze/thaw cycles lead to a progressive loss of structural and molecular integrity in deep frozen specimens. The aim of this study was to evaluate a micro-CT stage, which maintains the cryoconservation of large specimens throughout micro-CT imaging. Deep frozen ovine vertebral segments (-20 °C) were fixed in a micro-CT stage made of expanded polystyrene and cooled with dry ice (0 g, 60 g and 120 g). The temperature inside the stage was measured half-hourly over a time span of three hours with subsequent measurement of surface temperature. The method was validated in a series of 30 deep frozen vertebral specimens and in liver tissue after repetitive micro-CT scanning. Isolation without cooling resulted in defrosting. Cooling with 60 g of dry ice led to a temperature rise inside the stage (max. 5.1 °C) and on the specimen surfaces (max. -3 °C). Cooling with 120 g of dry ice resulted in a significant (p < 0.001) and sufficient lowering of the temperature inside the stage (max. -14 °C) and on the surface of the specimens (max. -13.9 °C). The surface temperature during the subsequent micro-CT validation study did not exceed -16 °C (processing time 1 h 45 min). The resolution was 33 µm isotropic voxel side length, enabling a binarization of bone microstructures. Temperature can reliably be maintained below -10 °C during a micro-CT scan by applying the described technique. The resulting spatial resolution and image quality permits a binarization of bone microstructure.