Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Genetic structure of phenotypic robustness in the collaborative cross mouse diallel panel.

Developmental stability and canalization describe the ability of developmental systems to minimize phenotypic variation in the face of stochastic micro-environmental effects, genetic variation and environmental influences. Canalization is the ability to minimize the effects of genetic or environmental effects, whereas developmental stability is the ability to minimize the effects of micro-environmental effects within individuals. Despite much attention, the mechanisms that underlie these two components of phenotypic robustness remain unknown. We investigated the genetic structure of phenotypic robustness in the collaborative cross (CC) mouse reference population. We analysed the magnitude of fluctuating asymmetry (FA) and among-individual variation of cranial shape in reciprocal crosses among the eight parental strains, using geometric morphometrics and a diallel analysis based on a Bayesian approach. Significant differences among genotypes were found for both measures, although they were poorly correlated at the level of individuals. An overall positive effect of inbreeding was found for both components of variation. The strain CAST/EiJ exerted a positive additive effect on FA and, to a lesser extent, among-individual variance. Sex- and other strain-specific effects were not significant. Neither FA nor among-individual variation was associated with phenotypic extremeness. Our results support the existence of genetic variation for both developmental stability and canalization. This finding is important because robustness is a key feature of developmental systems. Our finding that robustness is not related to phenotypic extremeness is consistent with theoretical work that suggests that its relationship to stabilizing selection is not straightforward.

Epigenetic interactions and the structure of phenotypic variation in the cranium.

Understanding the developmental and genetic basis for evolutionarily significant morphological variation in complex phenotypes such as the mammalian skull is a challenge because of the sheer complexity of the factors involved. We hypothesize that even in this complex system, the expression of phenotypic variation is structured by the interaction of a few key developmental processes. To test this hypothesis, we created a highly variable sample of crania using four mouse mutants and their wild-type controls from similar genetic backgrounds with developmental perturbations to particular cranial regions. Using geometric morphometric methods we compared patterns of size, shape, and integration in the sample within and between the basicranium, neurocranium, and face. The results highlight regular and predictable patterns of covariation among regions of the skull that presumably reflect the epigenetic influences of the genetic perturbations in the sample. Covariation between relative widths of adjoining regions is the most dominant factor, but there are other significant axes of covariation such as the relationship between neurocranial size and basicranial flexion. Although there are other sources of variation related to developmental perturbations not analyzed in this study, the patterns of covariation created by the epigenetic interactions evident in this sample may underlie larger scale evolutionary patterns in mammalian craniofacial form.

Trabecular bone in the bird knee responds with high sensitivity to changes in load orientation.

Wolff's law of trajectorial orientation proposes that trabecular struts align with the orientation of dominant compressive loads within a joint. Although widely considered in skeletal biology, Wolff's law has never been experimentally tested while controlling for ontogenetic stage, activity level, and species differences, all factors that may affect trabecular bone growth. Here we report an experimental test of Wolff's law using a within-species design in age-matched subjects experiencing physiologically normal levels of bone strain. Two age-matched groups of juvenile guinea fowl Numida meleagris ran on a treadmill set at either 0 degrees (Level group) or 20 degrees (Incline group), for 10 min per day over a 45-day treatment period. Birds running on the 20 degrees inclined treadmill used more-flexed knees than those in the Level group at midstance (the point of peak ground reaction force). This difference in joint posture enabled us to test the sensitivity of trabecular alignment to altered load orientation in the knee. Using a new radon transform-based method for measuring trabecular orientation, our analysis shows that the fine trabecular bone in the distal femur has a high degree of correspondence between changes in joint angle and trabecular orientation. The sensitivity of this response supports the prediction that trabecular bone adapts dynamically to the orientation of peak compressive forces.

Bisphosphonates reduce bone mineral loss at ligament entheses after joint injury.

OBJECTIVE: To examine the effects of anterior cruciate ligament (ACL) insufficiency, and subsequent bisphosphonate (BP) antiresorptive therapy, on the bone mineral interface at the enthesis of remaining ligamentous restraints. METHODS: We measured bone mineral geometry (and subsequent adaptation) at the medial collateral ligament (MCL) origin, using micro-computed tomography (muCT). Groups of normal control, 6 and 14 wk anterior cruciate ligament transected (ACLX), and 6 wk ACLX-BP (risedronate) dosed rabbits were evaluated. Samples were then processed histologically, and the results of mineral adaptation and progression of osteoarthritis (OA) compared to joint laxity values obtained from previous biomechanical testing of the MCL-complex. RESULTS: muCT defined the MCL origin as a symmetrical, metaphyseal depression that contained soft-tissue elements, including fibrocartilage and ligament--as seen in subsequent histological sections. In contrast, the insertions from ACLX animals lost significant bone mineral, with an MCL-insertion volume 1.2 times that of normal controls at 6 wk ACLX, which further increased to 2.3 times that of normal controls at 14 wk ACLX. Significant differences were also measured between 6 and 14 wk ACLX and age-matched normal controls in volume of cortical bone containing the MCL insertion. However, there were no significant differences in the percentage of cortical bone to underlying trabecular bone at the MCL insertion. When comparing muCT mineral adaptation at the MCL-enthesis with historical MCL-complex laxity data, the values for laxity after ACLX increased proportionately as bone mineral at the insertion was lost, and subsequent use of the BP risedronate reduced both mineral loss and MCL-complex laxity. CONCLUSION: Compared to the untreated ACLX condition, administering bisphosphonate immediately after loss of the ACL conserved bone mineral at the MCL enthesis, suggesting the potential to therapeutically influence joint-complex laxity and OA progression.

Comparison of microcomputed tomographic and microradiographic measurements of cortical bone porosity.

Cortical bone is perforated by a network of canals that have a significant impact upon its material properties. Microcomputed tomography offers the possibility of noninvasively visualizing and quantifying cortical pores in both two and three dimensions. Establishing how two-dimensional (2D) microcomputed tomographic (microCT) analysis compares with conventional methods for analyzing cortical porosity is an important prerequisite for the wider adoption of this technique and the development of three-dimensional (3D) analysis. Therefore, we compared porosity-related parameters from 2D microcomputed tomographic images with those from matching microradiographic sections. Samples from five human femora were scanned at a 10-microm resolution and then sequentially sectioned and microradiographed. An average of eight image pairs were produced from each femur (total, n = 41). The repeatability and comparability of the two techniques was assessed for three parameters; cortical porosity (%), mean pore area (microm(2)), and pore density (pores/mm(2)). For repeatability, no significant difference ( P > 0.05) was found between the two methods for cortical porosity and mean pore area; however, pore density differed significantly ( P < 0.001). For comparability, the bias (+/- error) between the methods was found to be 0.51% (+/-0.31%) for cortical porosity and -155 microm(2) (+/-293 microm(2)) for mean pore area. The bias for pore density was dependent upon measurement size with microcomputed tomographic images having 14% (+/-9.3%) fewer pores per millimeter squared. The qualitative and quantitative similarities between the two techniques demonstrated the utility of 2D microcomputed tomographic for cortical porosity analysis. However, the relatively poor results for pore density revealed that a higher resolution (<10 microm) is needed to consistently visualize all cortical pores in human bone.

Quantitative 3D analysis of the canal network in cortical bone by micro-computed tomography.

Cortical bone is perforated by an interconnected network of porous canals that facilitate the distribution of neurovascular structures throughout the cortex. This network is an integral component of cortical microstructure and, therefore, undergoes continual change throughout life as the cortex is remodeled. To date, the investigation of cortical microstructure, including the canal network, has largely been limited to the two-dimensional (2D) realm due to methodological hurdles. Thanks to continuing improvements in scan resolution, micro-computed tomography (muCT) is the first nondestructive imaging technology capable of resolving cortical canals. Like its application to trabecular bone, muCT provides an efficient means of quantifying aspects of 3D architecture of the canal network. Our aim here is to introduce the use of muCT for this application by providing examples, discussing some of the parameters that can be acquired, and relating these to research applications. Although several parameters developed for the analysis of trabecular microstructure are suitable for the analysis of cortical porosity, the algorithm used to estimate connectivity is not. We adapt existing algorithms based on skeletonization for this task. We believe that 3D analysis of the dimensions and architecture of the canal network will provide novel information relevant to many aspects of bone biology. For example, parameters related to the size, spacing, and volume of the canals may be particularly useful for investigation of the mechanical properties of bone. Alternatively, parameters describing the 3D architecture of the canal network, such as connectivity between the canals, may provide a means of evaluating cumulative remodeling related change.

Angiogenesis in the distal femoral chondroepiphysis of the rabbit during development of the secondary centre of ossification.

In the developing chondroepiphyses of long bones, the avascular cartilaginous anlage is invaded by numerous blood vessels, through the process of angiogenesis. The objective of this study was to investigate the chronology of this vascular invasion with the spontaneous calcification of the cartilaginous epiphysis during development of the secondary ossification centre in the rabbit distal femur. The time-course of chondroepiphyseal vascular invasion was determined histologically and standardized for eight gestational and four postnatal intervals by plotting kit body mass against crown-rump length. Similarly, microcomputed tomography (micro-CT) helped to visualize calcification at those same gestational and postnatal intervals. To confirm the angiogenic nature of the avascular chondroepiphysis, such samples were assayed on the chick chorio-allantoic membrane (CAM). Neovascular outgrowths from the CAM were apparent 48 h following introduction of an 18-day (gestational) chondroepiphyseal sample. Chondroepiphyseal samples were assayed for the potent developmental angiogenic factors bFGF and VEGF, with the mRNA expression for both these mediators being confirmed using RT-PCR. As angiogenesis and calcification during chondroepiphyseal development occur in a defined tissue environment initially devoid of blood vessels and mineral, those processes provided a unique opportunity to study their progression without complication of injury-related inflammation or extant vasculature and mineral. Furthermore, the discovery of angiogenic, angiostatic or mineral-regulating mediators specific to developing connective tissue may prove useful for analysing the regulation of vascular and mineral pathogenesis in articular tissues.