Leptin receptor gene deficiency minimally affects osseointegration in rats.

Metabolic syndrome represents a cluster of conditions such as obesity, hyperglycaemia, dyslipidaemia, and hypertension that can lead to type 2 diabetes mellitus and/or cardiovascular disease. Here, we investigated the influence of obesity and hyperglycaemia on osseointegration using a novel, leptin receptor-deficient animal model, the Lund MetS rat. Machined titanium implants were installed in the tibias of animals with normal leptin receptor (LepR+/+) and those harbouring congenic leptin receptor deficiency (LepR-/-) and were left to heal for 28 days. Extensive evaluation of osseointegration was performed using removal torque measurements, X-ray micro-computed tomography, quantitative backscattered electron imaging, Raman spectroscopy, gene expression analysis, qualitative histology, and histomorphometry. Here, we found comparable osseointegration potential at 28 days following implant placement in LepR-/- and LepR+/+ rats. However, the low bone volume within the implant threads, higher bone-to-implant contact, and comparable biomechanical stability of the implants point towards changed bone formation and/or remodelling in LepR-/- rats. These findings are corroborated by differences in the carbonate-to-phosphate ratio of native bone measured using Raman spectroscopy. Observations of hypermineralised cartilage islands and increased mineralisation heterogeneity in native bone confirm the delayed skeletal development of LepR-/- rats. Gene expression analyses reveal comparable patterns between LepR-/- and LepR+/+ animals, suggesting that peri-implant bone has reached equilibrium in healing and/or remodelling between the animal groups.

Transformation of bone mineral morphology: From discrete marquise-shaped motifs to a continuous interwoven mesh.

Continual bone apposition at the cranial sutures provides the unique opportunity to understand how bone is built. Bone harvested from 16-week-old Sprague Dawley rat calvaria was either (i) deproteinised to isolate the inorganic phase (i.e., bone mineral) for secondary electron scanning electron microscopy or (ii) resin embedded for X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and micro-Raman spectroscopy. Interdigitated finger-like projections form the interface between frontal and parietal bones. Viewed from the surface, bone mineral at the mineralisation front is comprised of nanoscale mineral platelets arranged into discrete, ~0.6-3.5 µm high and ~0.2-1.5 µm wide, marquise-shaped motifs that gradually evolve into a continuous interwoven mesh of mineralised bundles. Marquise-shaped motifs also contribute to the burial of osteoblastic-osteocytes by contributing to the roof over the lacunae. In cross-section, apices of the finger-like projections resemble islands of mineralised tissue, where new bone apposition at the surface is evident as low mineral density areas, while the marquise-shaped motifs appear as near-equiaxed assemblies of mineral platelets. Carbonated apatite content is higher towards the internal surface of the cranial vault. Up to 4 µm from the bone surface, strong Amide III, Pro, Hyp, and Phe signals, distinct PO4 3- bands, but negligible CO3 2- signal indicate recent bone formation and/or delayed maturation of the mineral. We show, for the first time, that the extracellular matrix of bone is assembled into micrometre-sized units, revealing a superstructure above the mineralised collagen fibril level, which has significant implications for function and mechanical competence of bone.

Mapping Bone Surface Composition Using Real-Time Surface Tracked Micro-Raman Spectroscopy.

The surface of bone tells a story - one that is worth a thousand words - of how it is built and how it is repaired. Chemical (i.e., composition) and physical (i.e., morphology) characteristics of the bone surface are analogous to a historical record of osteogenesis and provide key insights into bone quality. Analysis of bone chemistry is of particular relevance to the advancement of human health, cell biology, anthropology/archaeology, and biomedical engineering. Although scanning electron microscopy remains a popular and versatile technique to image bone across multiple length scales, limited chemical information can be obtained. Micro-Raman spectroscopy is a valuable tool for nondestructive chemical/compositional analysis of bone. However, signal integrity losses occur frequently during wide-field mapping of non-planar surfaces. Samples for conventional Raman imaging are, therefore, rendered planar through polishing or sectioning to ensure uniform signal quality. Here, we demonstrate ν1 PO43- and ν1 CO32- peak intensity losses where the sample surface and the plane of focus are offset by over 1-2 µm when underfocused and 2-3 µm when overfocused at 0.5-1 s integration time (15 mW, 633 nm laser). A technique is described for mapping the composition of the inherently irregular/non-planar surface of bone. The challenge posed by the native topology characteristic of this unique biological system is circumvented via real-time focus-tracking based on laser focus optimization by continuous closed-loop feedback. At the surface of deproteinized and decellularized/defatted sheep tibial cortical bone, regions of interest up to 1 mm2 were scanned at micrometer and submicrometer resolution. Despite surface height deviations exceeding 100 µm, it is possible to seamlessly probe local gradients in organic and inorganic constituents of the extracellular matrix as markers of bone metabolism and bone turnover, blood vessels and osteocyte lacunae, and the rope-like mineralized bundles that comprise the mineral phase at the bone surface.

50 years of scanning electron microscopy of bone-a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy.

Bone is an architecturally complex system that constantly undergoes structural and functional optimisation through renewal and repair. The scanning electron microscope (SEM) is among the most frequently used instruments for examining bone. It offers the key advantage of very high spatial resolution coupled with a large depth of field and wide field of view. Interactions between incident electrons and atoms on the sample surface generate backscattered electrons, secondary electrons, and various other signals including X-rays that relay compositional and topographical information. Through selective removal or preservation of specific tissue components (organic, inorganic, cellular, vascular), their individual contribution(s) to the overall functional competence can be elucidated. With few restrictions on sample geometry and a variety of applicable sample-processing routes, a given sample may be conveniently adapted for multiple analytical methods. While a conventional SEM operates at high vacuum conditions that demand clean, dry, and electrically conductive samples, non-conductive materials (e.g., bone) can be imaged without significant modification from the natural state using an environmental scanning electron microscope. This review highlights important insights gained into bone microstructure and pathophysiology, bone response to implanted biomaterials, elemental analysis, SEM in paleoarchaeology, 3D imaging using focused ion beam techniques, correlative microscopy and in situ experiments. The capacity to image seamlessly across multiple length scales within the meso-micro-nano-continuum, the SEM lends itself to many unique and diverse applications, which attest to the versatility and user-friendly nature of this instrument for studying bone. Significant technological developments are anticipated for analysing bone using the SEM.