Differential Gene Expression from Microarray Analysis Distinguishes Woven and Lamellar Bone Formation in the Rat Ulna following Mechanical Loading

Formation of woven and lamellar bone in the adult skeleton can be induced through mechanical loading. Although much is known about the morphological appearance and structural properties of the newly formed bone, the molecular responses to loading are still not well understood. The objective of our study was to use a microarray to distinguish the molecular responses between woven and lamellar bone formation induced through mechanical loading. Rat forelimb loading was completed in a single bout to induce the formation of woven bone (WBF loading) or lamellar bone (LBF loading). A set of normal (non-loaded) rats were used as controls. Microarrays were performed at three timepoints after loading: 1 hr, 1 day and 3 days. Confirmation of microarray results was done for a select group of genes using quantitative real-time PCR (qRT-PCR). The micorarray identified numerous genes and pathways that were differentially regulated for woven, but not lamellar bone formation. Few changes in gene expression were evident comparing lamellar bone formation to normal controls. A total of 395 genes were differentially expressed between formation of woven and lamellar bone 1 hr after loading, while 5883 and 5974 genes were differentially expressed on days 1 and 3, respectively. Results suggest that not only are the levels of expression different for each type of bone formation, but that distinct pathways are activated only for woven bone formation. A strong early inflammatory response preceded an increase in angiogenic and osteogenic gene expression for woven bone formation. Furthermore, at later timepoints there was evidence of bone resorption after WBF loading. In summary, the vast coverage of the microarray offers a comprehensive characterization of the early differences in expression between woven and lamellar bone formation

Histomorphometric evaluation of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial "critical size" defect

Objective: The aim of this study was to test specific stem cells that could enhance bone formation in combination with specific scaffolds. Methods: Dental Pulp Stem Cells (DPSCs) were seeded with Granular Deproteinized Bovine Bone (GDPB) or Beta-Tricalcium Phosphate (ß-TCP) in a rat model of calvarial "critical size" defect. DPSCs were isolated from permanent human teeth, obtained and characterized using specific stem cells markers (Nanog and Oct-4) by real time-PCR and immunofluorescence. Cells were differentiated for 10-15 days towards the osteoblastic phenotype with 100μM L-ascorbic acid, added every day in culture medium and 20 vol. percentage of FBS in α-MEM medium. Osteogenic commitment was evaluated with real time-PCR by measuring the expression of specific markers (osteonectin and runx2). When a sufficient cell number was obtained, DPSCs were trypsinized, washed in culture medium and seeded onto the GDPB and ß-TCP scaffold sat a density of 0.5-1×106 cells/scaffold. Two bilateral critical-size circular defects (5 mm diameter; 1 mm thickness) were created from the parietal bone of the 8 athymic T-cell deficient nude rats. One cranial defect for each rat was filled with the scaffold alone and the other defect with the scaffold seeded with stem cells. After 12 weeks post-surgery animals were euthanized and histomorphometric analysis was performed. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test. A p-value <0.05 was considered statistically significant. Results: GDPB group presented higher percentage of lamellar bone than that of GDPB/DPSC, ß-TCP alone had lower levels as compared to ß-TCP/DPSC. The addition of stem cells significantly increased woven bone formation in both scaffold-based implants, although still higher in GDPB based implants. Conclusion: Our findings indicate that GDPB and ß-TCP used as scaffold to induce bone regeneration may benefit from adding DPSC to tissue-engineered constructs

Synthesis and characterization of novel scaffold for bone tissue engineering based on Whartons´s jelly

A composite is a material made of more than one component, and the bond between the components is on a scale larger than the atomic scale. The objective of the present study was to synthesize and perform the structural characterization and biological evaluation of a new biocomposite (BCO) based on a novel combination of an organic and an inorganic phase, for bone tissue engineering applications. The organic phase consisted of Wharton´s Jelly (WJ), which was obtained from embryonic tissue following a protocol developed by our laboratory. The inorganic phase consisted of bioceramic particles (BC), produced by sintering hydroxyapatite (HA) with β- tricalcium phosphate (β-TCP), and bioactive glass particles (BG). Each phase of the BCO was fully characterized by SEM, EDS, XRD and FTIR. Biocompatibility was evaluated in vivo in the tibiae of Wistar rats (n=40). Histological evaluation was performed at 0, 1, 7, 14, 30 and 60 days. XRD showed the phases corresponding to HA and β-TCP, whereas diffractogram of BG showed it to have an amorphous structure. EDS showed mainly Si and Na, Ca, P in BG, and Ca and P in HA and β-TCP. FTIR identified bonds between the organic and inorganic phases. From a mechanical viewpoint, the composite showed high flexural strength of 40.3±0.8MPa. The synthesized BCO exhibited adequate biocompatibility as shown by formation of lamellar type bone linked by BG and BC particles. The biomaterial presented here showed excellent mechanical and biocompatibility properties for its potential clinical use.Fil: Martinez, Cristian. Universidad de Buenos Aires. Facultad de Ingenieria. Instituto de Ingeniería Biomédica; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Fernández, Carlos. Universidad de Buenos Aires. Facultad de Ingenieria. Instituto de Ingeniería Biomédica; ArgentinaFil: Prado, Miguel Oscar. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ozols, Andres. Universidad de Buenos Aires. Facultad de Ingenieria. Instituto de Ingeniería Biomédica; ArgentinaFil: Olmedo, Daniel Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentin

Hyperostotic tympanic bone spicules in domestic and wild animal species

Hyperostotic tympanic bone spicules (HTBS), or "mucoperiosteal exostoses" (ME, syn.) are small, globular (>= 1 mm in diameter), mostly stalked and drumstick-like, bony structures, which arise from the inner wall of the tympanic bulla and project into the middle ear cavity. HTBS present as mineral densities inside the tympanic bulla on radiographs or computed tomographic (CT) images. They have previously been referred to as "otoliths" and were thought to represent mineral concretions secondary to otitis media. Recently, it was shown that HTBS actually consist of regularly composed bone tissue, covered by normal middle ear mucosa. So far, HTBS have only extensively been described in dogs, where they occur with a prevalence of up to >45%. A recent study detected ME, most likely representing HTBS, in the tympanic cavities of skeletonised skull bones of African lions. To estimate the occurrence of HTBS in other mammal species, the middle ears of adult animals of 78 different domestic, wild, and zoo species undergoing routine necropsy at the Institute of Veterinary Pathology of the LMU Munich, Germany were examined in the present study. HTBS were found in the tympanic bullae of carnivorous species, such as canids (wolf, fox), and in several large felid species (lion, tiger, leopard, cheetah). In contrast, HTBS were not present in domestic cats (more than to 200 cases), small carnivorous species such as mustelids, nor in any primate, ungulate, ruminant, pig, insectivore, or rodent species. The detectability of HTBS by CT of the tympanic bullae of large felids was demonstrated in an African lion. Histologically, HTBS consisted of mature lamellar bone, covered by periosteum and a partially ciliated, flat epithelium, regularly without any apparent inflammatory alterations. The present study demonstrates that HTBS may frequently occur in large felids and in different canid species. These findings should be taken into account when examining the middle ear, or interpreting bulla radiographs/CT-images of the respective species. However, the factors triggering the development of HTBS remain to be identified

Osseointegration of zirconia implants compared with titanium : an in vivo study

Background Titanium and titanium alloys are widely used for fabrication of dental implants. Since the material composition and the surface topography of a biomaterial play a fundamental role in osseointegration, various chemical and physical surface modifications have been developed to improve osseous healing. Zirconia-based implants were introduced into dental implantology as an altenative to titanium implants. Zirconia seems to be a suitable implant material because of its tooth-like colour, its mechanical properties and its biocompatibility. As the osseointegration of zirconia implants has not been extensively investigated, the aim of this study was to compare the osseous healing of zirconia implants with titanium implants which have a roughened surface but otherwise similar implant geometries. Methods Forty-eight zirconia and titanium implants were introduced into the tibia of 12 minipigs. After 1, 4 or 12 weeks, animals were sacrificed and specimens containing the implants were examined in terms of histological and ultrastructural techniques. Results Histological results showed direct bone contact on the zirconia and titanium surfaces. Bone implant contact as measured by histomorphometry was slightly better on titanium than on zirconia surfaces. However, a statistically significant difference between the two groups was not observed. Conclusion The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants

3D Raman mapping of the collagen fibril orientation in human osteonal lamellae

AbstractChemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues

Histological evidence for a supraspinous ligament in sauropod dinosaurs

Supraspinous ossified rods have been reported in the sacra of some derived sauropod dinosaurs. Although different hypotheses have been proposed to explain the origin ofthis structure, histological evidence has never been provided to support or reject any of them. In order to establish its origin, we analyse and characterize the microstructure of thesupraspinous rod of two sauropod dinosaurs from the Upper Cretaceous of Argentina. The supraspinous ossified rod is almost entirely formed by dense Haversian bone. Remains ofprimary bone consist entirely of an avascular tissue composed of two types of fibre-like structures, which are coarse and longitudinally (parallel to the main axis of the element) oriented. These structures are differentiated on the basis of their optical properties under polarized light. Very thin fibrous strands are also observed in some regions. These small fibres are all oriented parallel to one another but perpendicular to the element main axis. Histological features of the primary bone tissue indicate that the sacral supraspinous rod corresponds to an ossified supraspinous ligament. The formation of this structure appears to have been a non-pathological metaplastic ossification, possibly induced by the continuous tensile forces applied to the element.Fil: Cerda, Ignacio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación en Paleobiología y Geología; Argentina. Universidad Nacional de Río Negro; ArgentinaFil: Casal, Gabriel. Universidad Nacional de la Patagonia; ArgentinaFil: Martínez, Rubén Darío. Universidad Nacional de la Patagonia ; ArgentinaFil: Ibiricu, Lucio Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentin

Investigations into the Ulnar Response to Mechanical Stimuli Activating Lamellar and Woven Bone Formation

Woven and lamellar bone formation can be stimulated using mechanical loading. Woven bone forms rapidly in response to damaging loading in a disorganized manner with low mineral density. In contrast, lamellar bone formation can be induced in the absence of damage, and is characterized by its slow, organized deposition and high density. In this dissertation, we first examined the molecular response to woven and lamellar bone formation using damaging and non-damaging dynamic loading protocols, respectively. We observed a significant increase in gene expression related to angiogenesis, cell proliferation and osteogenesis prior to woven bone formation, with significantly lower levels of expression associated with lamellar bone formation. To fully characterize the molecular responses of woven and lamellar bone we used a whole genome microarray. The micorarray results brought to light many inflammatory factors not previously investigated in our model, expanded previous findings about angiogenesis, and strengthened our understanding of the role of osteogenic pathways. Our investigations suggested that angiogenesis is required for successful woven bone formation. We used several angiogenic inhibitors, but were unable to prove the dependence of woven bone formation on angiogenesis. Finally, we sought to separate the effects of static and dynamic strains on bone formation. These findings demonstrate that in the absence dynamic strain, bone damage triggers a woven bone response that leads to a functional repair of whole-bone strength. Overall, the work done in this thesis has enhanced our understanding of bone formation. Future studies will expand on the microarray findings and clarify the role of angiogenesis in woven bone formation

Decision Tree Analysis as a Supplementary Tool to Enhance Histomorphological Differentiation when Distinguishing Human from Non-human Cranial Bone in both Burnt and Unburnt States: A feasibility study

This feasibility study was undertaken to describe and record the histological characteristics of burnt and unburnt cranial bone fragments from human and non-human bones. Reference series of fully mineralised, transverse sections of cranial bone, from all variables and specimen states were prepared by manual cutting and semi-automated grinding and polishing methods. A photomicrograph catalogue reflecting differences in burnt and unburnt bone from human and non-humans was recorded and qualitative analysis was performed using an established classification system based on primary bone characteristics. The histomorphology associated with human and non-human samples was, for the main part, preserved following burning at high temperature. Clearly, fibro-lamellar complex tissue subtypes, such as plexiform or laminar primary bone, were only present in non-human bones. A decision tree analysis based on histological features provided a definitive identification key for distinguishing human from non-human bone, with an accuracy of 100%. The decision tree for samples where burning was unknown was 96% accurate, and multi-step classification to taxon was possible with 100% accuracy. The results of this feasibility study, strongly suggest that histology remains a viable alternative technique if fragments of cranial bone require forensic examination in both burnt and unburnt states. The decision tree analysis may provide an additional, but vital tool to enhance data interpretation. Further studies are needed to assess variation in histomorphology taking into account other cranial bones, ontogeny, species and burning conditions