A robust methodology for the quantitative assessment of the rat jawbone microstructure.

Micro-computed tomography can be applied for the assessment of the micro-architectural characteristics of the cortical and trabecular bones in either physiological or disease conditions. However, reports often lack a detailed description of the methodological steps used to analyse these images, such as the volumes of interest, the algorithms used for image filtration, the approach used for image segmentation, and the bone parameters quantified, thereby making it difficult to compare or reproduce the studies. This study addresses this critical need and aims to provide standardized assessment and consistent parameter reporting related to quantitative jawbone image analysis. Various regions of the rat jawbones were screened for their potential for standardized micro-computed tomography analysis. Furthermore, the volumes of interest that were anticipated to be most susceptible to bone structural changes in response to experimental interventions were defined. In the mandible, two volumes of interest were selected, namely, the condyle and the trabecular bone surrounding the three molars. In the maxilla, the maxillary tuberosity region and the inter-radicular septum of the second molar were considered as volumes of interest. The presented protocol provides a standardized and reproducible methodology for the analysis of relevant jawbone volumes of interest and is intended to ensure global, accurate, and consistent reporting of its morphometry. Furthermore, the proposed methodology has potential, as a variety of rodent animal models would benefit from its implementation.

In vivo assessment of the effect of controlled high- and low-frequency mechanical loading on peri-implant bone healing.

The aim of this study was to investigate the effect of controlled high- (HF) and low-frequency (LF) mechanical loading on peri-implant bone healing. Custom-made titanium implants were inserted in both tibiae of 69 adult Wistar rats. For every animal, one implant was loaded by compression through the axis of tibia (test), whereas the other one was unloaded (control). The test implants were randomly distributed among four groups receiving different loading regimes, which were determined by ex vivo calibration. Within the HF (40 Hz) or LF (2 Hz) loading category, the magnitudes were chosen as low- (LM) and high-magnitude (HM), respectively, leading to constant strain rate amplitudes for the two frequency groups. This resulted in the four loading regimes: (i) HF-LM (40 Hz-0.5 N); (ii) HF-HM (40 Hz-1 N); (iii) LF-LM (2 Hz-10 N); and (iv) LF-HM (2 Hz-20 N) loading. Loading was performed five times per week and lasted for one or four weeks. Tissue samples were processed for histology and histomorphometry (bone-to-implant contact, BIC; and peri-implant bone fraction, BF) at the cortical and medullar level. Data were analysed statistically with ANOVA and paired t-tests with the significance level set at 0.05. For the one-week experiments, an increased BF adjacent to the implant surface at the cortical level was exclusively induced by the LF-HM loading regime (2 Hz-20 N). Four weeks of loading resulted in a significant effect on BIC (and not on BF) in case of HF-LM loading (40 Hz-0.5 N) and LF-HM loading (2 Hz-20 N): BIC at the cortical level significantly increased under both loading regimes, whereas BIC at the medullar level was positively influenced only in case of HF-LM loading. Mechanical loading at both HF and LF affects osseointegration and peri-implant BF. Higher loading magnitudes (and accompanying elevated tissue strains) are required under LF loading to provoke a positive peri-implant bone response, compared with HF loading. A sustained period of loading at HF is needed to result in an overall enhanced osseointegration.

Establishment of an in vivo model for molecular assessment of titanium implant osseointegration in compromised bone.

Shortening of the healing time before loading risks impeding successful titanium implant anchorage into compromised bone. A thorough understanding at the genetic scale of the early phases of bone regeneration at the implant interface is required before the development of strategies to enhance implant osseointegration. In this study a new in vivo implant model to explore the mechanism by which titanium implant osseointegration is affected by the host bone properties is presented. An implant was conceptualized enabling standardized harvesting of peri-implant tissue for quantitative molecular analysis while preserving the mimicking of the clinical setting. The implant is partly indented to provide a well-defined healing compartment from where tissue differentiation and de novo bone formation can be investigated and partly screw-threaded to provide a good implant anchorage into the bone. The feasibility of the implant design was assessed in osteopenic bone conditions, evoked by simulated weightlessness. Wistar rats were either hindlimb unloaded by tail suspension (HU) for 9 days or acted as controls (CTL). The status of compromised bone tissue through 9-days HU was confirmed by micro-X-ray computed tomography. The implant was installed in the proximal tibial bone 7 days after the onset of HU or CTL. Two days postimplantation, the peri-implant regenerating tissue responses were recorded by measuring expression of inflammatory, angiogenic, and bone resorption parameters (hypoxia-inducible factor 1, alpha subunit; vascular endothelial growth factor A; angiopoietin 1; endothelial PAS domain protein 1; fibroblast growth factor 2; tumor necrosis factor; interleukin 11; acid phosphatase 5, tartrate resistant; tumor necrosis factor (ligand) superfamily, member 11/RANKL). We successfully demonstrated that HU-associated bone conditions evoked a significant alteration of expression of the angiogenic markers in the peri-implant regenerative tissue during initial implant osseointegration, whereas the expression levels of the inflammatory and bone resorption parameters remained unchanged. We concluded that this in vivo implant model provides a well-designed and controlled method to examine molecular responses in implant osseointegration to impaired bone conditions. This model may serve to explore the application of anabolic strategies in peri-implant osteogenesis.