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Simple Summary: Before calcification, the early skeletal development of both Homo sapiens and the chondrichthyan fish Raja asterias is exclusively cartilaginous. This cartilage is formed through tissue segmentation and chondrocyte mitoses. Mineral deposition in the cartilage matrix influences the stiffness and shape of the skeletal segments. In mammals, calcified cartilage serves as a scaffold for bone deposition, which is then remodeled. Conversely, chondrichthyans retain calcified cartilage as their skeletal structure, forming calcification nuclei or "tesserae". These structures adapt to aquatic locomotion. In mammals, endochondral ossification provides limb bones with the necessary stiffness for terrestrial locomotion. X-rays of marine mammals show how endochondral ossification in dolphin flippers adapts to aquatic demands, including shortening of certain bones and an increase in elements in the autopodium's central rays. Before calcification begins, the early embryonic and fetal skeletal development of both mammalian Homo sapiens and the chondrichthyan fish Raja asterias consists exclusively of cartilage. This cartilage is formed and shaped through processes involving tissue segmentation and the frequency, distribution, and orientation of chondrocyte mitoses. In the subsequent developmental phase, mineral deposition in the cartilage matrix conditions the development further. The stiffness and structural layout of the mineralized cartilage have a significant impact on the shape of the anlagen (early formative structure of a tissue, a scaffold on which the new bone is formed) and the mechanical properties of the skeletal segments. The fundamental difference between the two studied species lies in how calcified cartilage serves as a scaffold for osteoblasts to deposit bone matrix, which is then remodeled. In contrast, chondrichthyans retain the calcified cartilage as the definitive skeletal structure. This study documents the distinct mineral deposition pattern in the cartilage of the chondrichthyan R. asterias, in which calcification progresses with the formation of focal calcification nuclei or "tesserae". These are arranged on the flat surface of the endo-skeleton (crustal pattern) or aligned in columns (catenated pattern) in the radials of the appendicular skeleton. This anatomical structure is well adapted to meet the mechanical requirements of locomotion in the water column. Conversely, in terrestrial mammals, endochondral ossification (associated with the remodeling of the calcified matrix) provides limb bones with the necessary stiffness to withstand the strong bending and twisting stresses of terrestrial locomotion. In this study, radiographs of marine mammals (reproduced from previously published studies) document how the endochondral ossification in dolphin flippers adapts to the mechanical demands of aquatic locomotion. This adaptation includes the reduction in the length of the stylopodium and zeugopodium and an increase in the number of elements in the autopodium's central rays. [ABSTRACT FROM AUTHOR]

Scanning electron microscopy (SEM) is a precious tool in materials science and morphology sciences, enabling detailed examination of materials at the nanoscale. However, precise and accurate sample repositioning during different observation sessions remains a significant challenge, impacting the quality and repeatability of SEM analyses. This study aimed to develop and evaluate a LEGO®‐based sample positioning system for SEM analysis. The system was designed to consistently identify and align features across multiple repositioning cycles, maintain accurate positioning along the z‐axis, minimize distortion, and provide repeatable and reliable results. The results indicated a high degree of precision and accuracy in the repositioning process, as evidenced by the minimal displacements, deviations in scaling and shearing, and the highly significant results (p < 0.001) obtained from the analysis of absolute translations and rotations. Moreover, the analyses were consistently replicated across six repetitions, underscoring the reliability of the observed results. While the findings suggest that the LEGO‐based sample positioning system is promising for enhancing SEM analyses' quality and repeatability, further studies are needed to optimize the system's design and evaluate its performance in different SEM applications. Ultimately, this study contributes to the ongoing efforts to develop cost‐effective, customizable, and accurate solutions for sample positioning in SEM, contributing to the advancement of materials science research and all SEM analysis requiring overtime observations of the same sample. Research Highlights: This study focused on the development and evaluation of a novel LEGO‐based sample positioning system specifically designed for SEM analysis.One of the standout features of this system is its ability to consistently identify and align features across multiple repositioning cycles, showcasing its precision and reliability.To further understand the mechanical aspects of the SEM stage, we employed the Rambold Kontroll comparator, which provided a baseline understanding of its mechanical tolerance.The registration process results were particularly noteworthy, as they revealed high accuracy with minimal displacements.Furthermore, the consistent outcomes observed across multiple repetitions emphasize the reliability and robustness of the methods we employed in this research. [ABSTRACT FROM AUTHOR]

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un‐remodeled throughout the life span. Ca2+ and PO43− concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system. Highlights: Comparative physico‐chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices.R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values.Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix.These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs.Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix. [ABSTRACT FROM AUTHOR]

We investigated the interfaces of the epiphyseal plate with over‐ and underlying bone segments using an integrated approach of histochemistry, microtomography and scanning electron microscopy (SEM) to overcome the inherent limitations of sections‐based techniques. Microtomography was able to provide an unobstructed, frontal view of large expanses of the two bone surfaces facing the growth plate, while SEM observation after removal of the soft matrix granted an equally unhindered access with a higher resolution. The two interfaces appeared widely dissimilar. On the diaphyseal side the hypertrophic chondrocytes were arranged in tall columns packed in a sort of compact palisade; the interposed extracellular matrix was actively calcifying into a thick mineralized crust growing towards the epiphysis. Behind the mineralization front, histochemical data revealed a number of surviving cartilage islets which were being slowly remodelled into bone. In contrast, the epiphyseal side of the cartilage consisted of a relatively quiescent reserve zone whose mineralization was marginal in amount and discontinuous in extension; the epiphyseal bone consisted of a loose trabecular meshwork, with ample vascular spaces opening directly into the non‐mineralized cartilage. On both sides the calcification process took place through the formation of spheroidal bodies 1–2 μm wide which gradually grew by apposition and coalesced into a solid mass, in a way distinctly different from that of bone and other calcified tissues. [ABSTRACT FROM AUTHOR]

The macroscopic and microscopic morphology of the appendicular skeleton was studied in the two species Raja asterias (order Rajiformes) and Torpedo marmorata (Order Torpediniformes), comparing the organization and structural layout of pectoral, pelvic, and tail fin systems. The shape, surface area and portance of the T. marmorata pectoral fin system (hydrodynamic lift) were conditioned by the presence of the two electric organs in the disk central part, which reduced the pectoral fin surface area, suggesting a lower efficiency of the "flapping effectors" than those of R. asterias. Otherwise, radials' rays alignment, morphology and calcification pattern showed in both species the same structural layout characterized in the fin medial zone by stiffly paired columns of calcified tiles in the perpendicular plane to the flat batoid body, then revolving and in the horizontal plane to continue as separate mono‐columnar rays in the fin lateral zone with a morphology suggesting fin stiffness variance between medial/lateral zone. Pelvic fins morphology was alike in the two species, however with different calcified tiles patterns of the 1st compound radial and pterygia in respect to the fin‐rays articulating perpendicularly to the latter, whose tile rows lay‐out was also different from that of the pectoral fins radials. The T. marmorata tail‐caudal fin showed a muscular and connective scaffold capable of a significant oscillatory forward thrust. On the contrary, the R. asterias dorsal tail fins were stiffened by a scaffold of radials‐like calcified segments. Histomorphology, heat‐deproteination technique and morphometry provided new data on the wing‐fins structural layout which can be correlated to the mechanics of the Batoid swimming behavior and suggested a cartilage‐calcification process combining interstitial cartilage growth (as that of all vertebrates anlagen) and a mineral deposition with accretion of individual centers (the tiles). The resulting layout showed scattered zones of un‐mineralized matrix within the calcified mass and a less compact texture of the matrix calcified fibers suggesting a possible way of fluid diffusion throughout the mineralized tissue. These observations could explain the survival of the embedded chondrocytes in absence of a canalicular system as that of the cortical bone. [ABSTRACT FROM AUTHOR]

Among all strategies directed at developing new tools to support re-vascularization of damaged tissues, the use of pro-angiogenic soluble factors, derived from mesenchymal stem cells (MSCs), appears a promising approach for regenerative medicine. Here, we compared the feasibility of two devices, generated by coupling soluble factors of human dental pulp mesenchymal stem cells (DPSCs), with a nanostructured scaffold, to support angiogenesis once transplanted in mice. DPSCs were obtained from impacted wisdom tooth removal, usually considered surgical waste material. After 28 days, we verified the presence of active blood vessels inside the scaffold through optical and scansion electron microscopy. The mRNA expression of surface antigens related to macrophage polarization (CD68, CD80, CD86, CD163, CD206), as well as pro-angiogenic markers (CD31, CD34, CD105, Angpt1, Angpt2, CDH5) was evaluated by real-time PCR. Our results demonstrate the capability of DPSC–scaffold and DPSC soluble factors–scaffold to support angiogenesis, similarly to adipose stem cells, whereas the absence of blood vessels was found in the scaffold grafted alone. Our results provide evidence that DPSC-conditioned medium can be proposed as a cell-free preparation able to support angiogenesis, thus, providing a relevant tool to overcome the issues and restrictions associated with the use of cells. [ABSTRACT FROM AUTHOR]

This scientific article investigates the mechanical properties of a novel three-dimensional-printing resin specifically designed for orthodontic aligners and compares it to other commonly used resins. The resin was made by blending two commercially available and certified resins to produce aligners with improved mechanical properties compared to each separate resin. The study examined the effect of the addition of NextDent Ortho Rigid resin on the mechanical properties of the specimens, more specifically analyzing the relationship between the amount of the added rigid resin and the mechanical properties of the specimens. The mechanical properties of the specimens and the nickel-titanium wires were analyzed using a three-point bending test setup. The results showed no statistically significant differences within the different groups of specimens. The study provides valuable insights into the potential of the resin to meet the mechanical demands of orthodontic treatments. It will contribute to the advancement of personalized orthodontic care through three-dimensional-printing technology. Blending commercially available and certified resins enables orthodontic practitioners to select the most suitable resin for each case, providing better outcomes for patients and increasing the efficiency of the treatment process. Further research and evaluation would be required to determine the suitability of the blended resin for orthodontic treatments. [ABSTRACT FROM AUTHOR]

The relationship between cartilage growth – mineralization patterns were studied in adult Rajidae with X‐ray morphology/morphometry, undecalcified resin‐embedded, heat‐deproteinated histology and scanning electron microscopy. Morphometry of the wing‐fins, nine central rays of the youngest and oldest specimens documented a significant decrement of radials mean length between inner, middle and outer zones, but without a regular progression along the ray. This suggests that single radial length growth is regulated in such a way to align inter‐radial joints parallel to the wing metapterygia curvature. Trans‐illumination and heat‐deproteination techniques showed polygonal and cylindrical morphotypes of tesserae, whose aligned pattern ranged from mono‐columnar, bi‐columnar, and multi‐columnar up to the crustal‐like layout. Histology of tessellated cartilage allowed to identify of zones of the incoming mineral deposition characterized by enhanced duplication rate of chondrocytes with the formation of isogenic groups, whose morphology and topography suggested a relationship with the impending formation of the radials calcified column. The morphotype and layout of radial tesserae were related to mechanical demands (stiffening) and the size/mass of the radial cartilage body. The cartilage calcification pattern of the batoids model shares several morphological features with tetrapods' endochondral ossification, that is, (chondrocytes' high duplication rate, alignment in rows, increased volume of chondrocyte lacunae), but without the typical geometry of the metaphyseal growth plates. Research Highlights: 1. The wing‐fins system consists of stiff radials, mobile inter‐radial joints and a flat inter‐radial membrane adapted to the mechanical demand of wing wave movement. 2. Growth occurs by forming a mixed calcified‐uncalcified cartilage texture, developing intrinsic tensional stresses documented by morphoanatomical data. [ABSTRACT FROM AUTHOR]

The authors studied the morphology of the upper and lower jaws, vertebrae and dorsal‐fin rays of the teleost fish Xiphias gladius to analyse the skeletal architecture and ossification pattern. The analogies and differences among these segments were investigated to identify a common morphogenetic denominator of the bone tissue osteogenesis and modeling. The large fat glands in the proximal upper jaw and their relationship to the underlying cartilage (absent in the lower jaw) suggested that there is a mechanism that explains rostral overgrowth in the Xiphiidae and Istiophoriidae families. Thus far, the compact structure of the distal rostrum has been interpreted as being the result of remodeling. Nonetheless, no evidence of cutting cones, scalloped outer border of osteons and sequence of bright–dark bands in polarized light was observed in this study, suggesting a primary osteon texture formed by compacting of collagen matrix and mineral deposition in the fat stroma lacunae of the bone, but without being oriented in layers of the collagen fibrils. A similar histology also characterizes the circular structures present in the other examined segments of the skeleton. The early phases of fibrillogenesis carried out by fibroblast‐like cells occurred farther from the already‐calcified bone surface inside the fat stroma lacunae. The fibrillar matrix was compacted and underwent mineral deposition near the previously calcified bone surface. This pattern of collagen matrix synthesis and calcification was different from that of mammalian osteoblasts, especially concerning the ability to build a lacuno‐canalicular system among cells. Necrosis or apoptosis of the latter and refilling of the empty lacunae by mineral deposits might explain the anosteocytic bone formation. [ABSTRACT FROM AUTHOR]

The skeleton of the batoid fish consists of a mixture of calcified and uncalcified cartilage with a typical layout of mineral deposition toward the outer border, leaving an uncalcified central core in most of the skeleton segments. An exception is observed in the radials, where mineral deposition is central. Joints and endoskeleton segments were studied in two adult samples of Raja cf. polystigma. Histomorphology, mineral deposition pattern, and zonal chondrocyte duplication activity were compared among several endoskeleton segments, but with particular attention to the fin rays; in the first, the uncalcified cartilage is central with an outer layer ranging from mineralized tesserae to a continuous calcified coating, whereas in the second, the uncalcified cartilage surrounds one or more central calcified columns. The diarthroses have a joint cavity closed by a fibrous capsule and the sliding surfaces rest on the base of mineralized tesserae, whereas the interradial amphiarthroses show a layer of densely packed chondrocytes between the flat, calcified discs forming the base of neighboring radials. In the endoskeleton segments, three types of tesserae are distinguished, characterizing the phases of skeletal growth and mineralization which present differences in each endoskeleton segment. The chondrocyte density between central core, subtesseral layer, and radial external cartilage did not show significant differences, while there was a significant difference in chondrocyte density between the latter zones and the type c tesserae of the pelvic girdle. The histomorphology and morphometry observed in Raja cf. polystigma suggest a model of cartilage growth associated with structural stiffening without remodeling. A key point of this model is suggested to be the incomplete mineralization of the tesseral layer and the continuous growth of cartilage, both enabling fluid diffusion through the matrix fibril network of scattered, uncalcified cartilage zones inside and between the tesserae. [ABSTRACT FROM AUTHOR]

The free surface of the articular cartilage must withstand compressive and shearing forces, maintain a low friction coefficient and allow oxygen and metabolites to reach the underlying matrix. In many ways it is critical to the physiology of the whole tissue and its disruption always involves deep pathological alterations and loss of the joint integrity. Being very difficult to image with section‐based conventional techniques, it was often described by previous research in conflicting terms or entirely overlooked. High‐magnification face‐on observations with high resolution scanning electron microscopy and with scanning probe microscopy revealed a very thin, delicate superficial layer rich in glycoconjugates, which may explain the very low friction coefficient of the tissue but which was very easily altered and/or dissolved in the preparation. Beneath this superficial sheet lies a thicker coat of thin, highly uniform, slightly wavy collagen fibrils lying parallel to the surface and mutually interconnected by a huge number of interfibrillar glycosaminoglycan bridges. These bridges and the collagen fibrils form an extended reticular structure able to redistribute tensile and compressive stress across a larger area of the surface and hence a greater volume of tissue. [ABSTRACT FROM AUTHOR]

Laser scalpels used in medical surgery concentrate light energy, heating the tissues. Recently, we reported thermoluminescence emission from laser‐treated soft tissues. Here we investigated the thermo‐optical effects caused by a laser operating at 808 nm on animal bones (beef ribs) through luminescence and fluorescence imaging, thermal imaging and scanning electron microscopy. Laser‐induced artificial lesions emitted luminescence peaking around 650 nm, with a half‐life of almost 1 hour. As concerns fluorescence, 24 hours after laser treatment we observed an increase of the emission and a shift from 500 (untreated) to 580 nm (treated). Recrystallization observed by SEM indicates that the temperature in the artificial lesions is over 600°C. We can conclude that laser treatment induces specific luminescent and fluorescent emissions due to heating of the bone and modification of its components. Monitoring these emissions could help prevent tissue overheating and its potential damages during laser‐assisted medical procedures. [ABSTRACT FROM AUTHOR]

Direct laser metal forming (DLMF) is a procedure in which a high-power laser beam is directed on a metal powder bed and programmed to fuse particles according to a computer-aided design file, thus generating a thin metal layer. With DLMF, it is now possible to fabricate dental implants with a superficial porous surface. The aim of the present study was to evaluate the peri-implant soft tissues around human-retrieved DLMF dental implants. Collagen fibers, in the form of bundles, were oriented perpendicularly to a distance of 100 μm from the surface, where they became parallel, running in several directions. In some portions, only a few collagen fiber bundles appeared to be oriented perpendicularly or obliquely to the plane of the section. Collagen fibers appeared to form a dense chaotic three-dimensional network running in different, more or less parallel directions to the surface. Under scanning electron microscopy, an intimate contact of the fibrous matrix with the implant surface was evident, and some collagen bundles could be seen to bind directly to the metal surface. By changing the surface microtexture, it was possible to change the response of the peri-implant soft tissues. [ABSTRACT FROM AUTHOR]

The aim of the present study was a light and scanning electron microscopy (SEM) evaluation of the peri-implant tissues around sandblasted, acid-etched implants, retrieved from man, after a loading period of 5 and 10 years, respectively. Two implants (Leone Implant System) had been retrieved for a fracture of the prosthetic superstructure respectively after 5 and 10 years of loading. Both implants were stable before retrieval and had been retrieved using a 5-mm trephine bur. One implant was treated to obtain thin ground sections, while the other underwent evaluation under SEM. Compact, mature lamellar bone was present over most of the implant perimeter in close contact with the implant surface and with many remodeling areas. Under SEM, small concavities, completely filled by mineralized bone, were present on the implant surface. The present histologic results showed that these implants were well integrated over the long term, and the peri-implant bone was undergoing continuous remodeling at the interface. [ABSTRACT FROM AUTHOR]

Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400–1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline‐like structure of tightly‐packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM‐EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not‐collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination.Complete crystalline transformation at 1200°C.Up to 15 days of decalcification fibrils period maintained. [ABSTRACT FROM AUTHOR]

The 'fate' of growth plate hypertrophic chondrocytes has been long debated with two opposing theories: cell apoptosis or survival with transformation into osteogenic cells. This study was carried out on the proximal tibial growth plate of rabbits using light microscopy, scanning and transmission electron microscopy. We focused particularly on the orientation of the specimens included in order to define the mineral deposition and the vascular invasion lines and obtain histological and ultrastructural images at the corresponding height of the plate. Chondrocyte morphology transformation through the maturation process (characterized by vesicles and then large cytoplasmic lacunae before condensation, fragmentation and disappearance of the nuclear chromatin) did not correspond to that observed in the 'in vitro' apoptosis models. These findings rather suggested the passage of free water from the cartilage matrix into a still live cell (swelling). The level of these changes suggested a close relationship with the mineral deposition line. Furthermore, the study provided evidence that the metaphyseal capillaries could advance inside the columns of stacked hypertrophic chondrocytes (delimited by the intercolumnar septa) without the need for calcified matrix resorption because the thin transverse septa between the stacked chondrocyte (below the mineral deposition line) were not calcified. The zonal distribution of cell types (hypertrophic chondrocytes, osteoblasts, osteoclasts and macrophages) did not reveal osteoclasts or chondroclasts at this level. Morphological and morphometric analysis recorded globular masses of an amorphous, necrotic material in a zone 0–70 μm below the vascular invasion line occasionally surrounded by a membrane (indicated as 'hypertrophic chondrocyte ghosts'). These masses and the same material not bound by a membrane were surrounded by a large number of macrophages and other blood cell precursors, suggesting this could be the cause of macrophage recall and activation. The most recent hypotheses based on genetic and lineage tracing studies stating that hypertrophic chondrocytes can survive and transform into osteoblasts and osteocytes (trans‐differentiation) were not confirmed by the ultrastructural morphology or by the zonal comparative counting and distribution of cell types below the vascular invasion line. [ABSTRACT FROM AUTHOR]

Purpose. The aim of this scanning electron microscope (SEM) study was to investigate the interface between the bone and a novel nanostructured calcium-incorporated dental implant surface in humans. Methods. A dental implant (Anyridge®, Megagen Implant Co., Gyeongbuk, South Korea) with a nanostructured calcium-incorporated surface (Xpeed®, Megagen Implant Co., Gyeongbuk, South Korea), which had been placed a month earlier in a fully healed site of the posterior maxilla (#14) of a 48-year-old female patient, and which had been subjected to immediate functional loading, was removed after a traumatic injury. Despite the violent trauma that caused mobilization of the fixture, its surface appeared to be covered by a firmly attached, intact tissue; therefore, it was subjected to SEM examination. The implant surface of an unused nanostructured calcium-incorporated implant was also observed under SEM, as control. Results. The surface of the unused implant showed a highly-structured texture, carved by irregular, multi-scale hollows reminiscent of a fractal structure. It appeared perfectly clean and devoid of any contamination. The human specimen showed trabecular bone firmly anchored to the implant surface, bridging the screw threads and filling the spaces among them. Conclusions. Within the limits of this human histological report, the sample analyzed showed that the nanostructured calcium-incorporated surface was covered by new bone, one month after placement in the posterior maxilla, under an immediate functional loading protocol. [ABSTRACT FROM AUTHOR]