Enhancing SEM positioning precision with a LEGO®-based sample fitting system.

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.

Human Dental Pulp Mesenchymal Stem Cell-Derived Soluble Factors Combined with a Nanostructured Scaffold Support the Generation of a Vascular Network In Vivo.

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.

Investigating the interfaces of the epiphyseal plate: An integrated approach of histochemistry, microtomography and SEM.

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.

Skeletal calcification patterns of batoid, teleost, and mammalian models: Calcified cartilage versus bone matrix.

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 PO4 3- concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2 O 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.

Adipose mesenchymal stem cell-derived soluble factors, produced under hypoxic condition, efficiently support in vivo angiogenesis.

Tissue regeneration or healing both require efficient vascularization within a tissue-damaged area. Based on this concept, a remarkable number of strategies, aimed at developing new tools to support re-vascularization of damaged tissue have emerged. Among the strategies proposed, the use of pro-angiogenic soluble factors, as a cell-free tool, appears as a promising approach, able to overcome the issues concerning the direct use of cells for regenerative medicine therapy. Here, we compared the effectiveness of adipose mesenchymal stem cells (ASCs), use as cell suspension, ASC protein extract or ASC-conditioned-medium (i.e., soluble factors), combined with collagenic scaffold, in supporting in vivo angiogenesis. We also tested the capability of hypoxia in increasing the efficiency of ASC to promote angiogenesis, via soluble factors, both in vivo and in vitro. In vivo studies were performed using the Integra® Flowable Wound Matrix, and the Ultimatrix in sponge assay. Flow cytometry was used to characterize the scaffold- and sponge-infiltrating cells. Real-time PCR was used to evaluate the expression of pro-angiogenic factors by stimulating Human Umbilical-Vein Endothelial Cells with ASC-conditioned media, obtained in hypoxic and normoxic conditions. We found that, in vivo, ACS-conditioned media can support angiogenesis similar to ASCs and ASC protein extract. Also, we observed that hypoxia increases the pro-angiogenic activities of ASC-conditioned media, compared to normoxia, by generating a secretome enriched in pro-angiogenic soluble factors, with bFGF, Adiponectine, ENA78, GRO, GRO-a, and ICAM1-3, as most regulated factors. Finally, ASC-conditioned media, produced in hypoxic condition, induce the expression of pro-angiogenic molecules in HUVECs. Our results provide evidence that ASC-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.

Fin systems comparative anatomy in model Batoidea Raja asterias and Torpedo marmorata: Insights and relatioships between musculo-skeletal layout, locomotion and morphology.

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.

The chondro-osseous junction of articular cartilage.

In the synovial joints the transition between the soft articular cartilage and the subchondral bone is mediated by a layer of calcified cartilage of structural and mechanical characteristics closer to those of bone. This layer, buried in the depth of articular cartilage, is not directly accessible and is mostly visualized in histological sections of decalcified tissue, where it appears as a darker strip in contact with the subchondral bone. In this study conventional histology and scanning electron microscopy (SEM) with secondary electron imaging (SE) or backscattered electron imaging (BSE) were used to discriminate the calcified and the uncalcified cartilage in high resolution on native, untreated tissue as well as in deproteinated or demineralized tissue. This approach evidenced a high heterogeneity of the calcified layer of articular cartilage. High resolution pictures revealed that the mineralization process originates by progressive accretion and confluence of individual, small mineral clusters, in a very different way from other hard tissues such as bone, dentin and mineralized tendons. Finally, selective removal of the soft matrix by thermal treatment allowed for the first time a face-on, unrestricted 3D view of the mineralization front.

The combined cartilage growth - calcification patterns in the wing-fins of Rajidae (Chondrichthyes): A divergent model from endochondral ossification of tetrapods.

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.

The complex rostral morphology and the endoskeleton ossification process of two adult samples of Xiphias gladius (Xiphiidae).

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.

Morphology of joints and patterns of cartilage calcification in the endoskeleton of the batoid Raja cf. polystigma.

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.

The Italian law on body donation: A position paper of the Italian College of Anatomists.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.

Case Report: Histological and Histomorphometrical Results of a 3-D Printed Biphasic Calcium Phosphate Ceramic 7 Years After Insertion in a Human Maxillary Alveolar Ridge.

Introduction: Dental implant placement can be challenging when insufficient bone volume is present and bone augmentation procedures are indicated. The purpose was to assess clinically and histologically a specimen of 30%HA-60%ß-TCP BCP 3D-printed scaffold, after 7-years. Case Description: The patient underwent bone regeneration of maxillary buccal plate with 3D-printed biphasic-HA block in 2013. After 7-years, a specimen of the regenerated bone was harvested and processed to perform microCT and histomorphometrical analyses. Results: The microarchitecture study performed by microCT in the test-biopsy showed that biomaterial volume decreased more than 23% and that newly-formed bone volume represented more than 57% of the overall mineralized tissue. Comparing with unloaded controls or peri-dental bone, Test-sample appeared much more mineralized and bulky. Histological evaluation showed complete integration of the scaffold and signs of particles degradation. The percentage of bone, biomaterials and soft tissues was, respectively, 59.2, 25.6, and 15.2%. Under polarized light microscopy, the biomaterial was surrounded by lamellar bone. These results indicate that, while unloaded jaws mimicked the typical osteoporotic microarchitecture after 1-year without loading, the BCP helped to preserve a correct microarchitecture after 7-years. Conclusions: BCP 3D-printed scaffolds represent a suitable solution for bone regeneration: they can lead to straightforward and less time-consuming surgery, and to bone preservation.

Biphasic Calcium Phosphate Biomaterials: Stem Cell-Derived Osteoinduction or In Vivo Osteoconduction? Novel Insights in Maxillary Sinus Augmentation by Advanced Imaging.

Maxillary sinus augmentation is often necessary prior to implantology procedure, in particular in cases of atrophic posterior maxilla. In this context, bone substitute biomaterials made of biphasic calcium phosphates, produced by three-dimensional additive manufacturing were shown to be highly biocompatible with an efficient osteoconductivity, especially when combined with cell-based tissue engineering. Thus, in the present research, osteoinduction and osteoconduction properties of biphasic calcium-phosphate constructs made by direct rapid prototyping and engineered with ovine-derived amniotic epithelial cells or amniotic fluid cells were evaluated. More in details, this preclinical study was performed using adult sheep targeted to receive scaffold alone (CTR), oAFSMC, or oAEC engineered constructs. The grafted sinuses were explanted at 90 days and a cross-linked experimental approach based on Synchrotron Radiation microCT and histology analysis was performed on the complete set of samples. The study, performed taking into account the distance from native surrounding bone, demonstrated that no significant differences occurred in bone regeneration between oAEC-, oAFMSC-cultured, and Ctr samples and that there was a predominant action of the osteoconduction versus the stem cells osteo-induction. Indeed, it was proven that the newly formed bone amount and distribution decreased from the side of contact scaffold/native bone toward the bulk of the scaffold itself, with almost constant values of morphometric descriptors in volumes more than 1 mm from the border.

Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta.

Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.

Photon emission and changes in fluorescent properties of bone after laser irradiation.

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.

Zooming on light packaging waste differences by scanning electron microscopy.

In the present paper, different types of pure and commercial plastic waste from different EU countries (UK, France, Italy, and Romania) were investigated for microstructure surface morphology and chemical properties by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDXS). The goal of the current investigation was to determine the chemical composition of selected packaging materials and compare these measurements with data obtained through a carbon-hydrogen-nitrogen-sulfur-oxygen (CHNS-O) elemental analyzer, which is conventionally used to characterize waste materials. The capabilities of the experimental approach are discussed in connection with their application to the study of waste sample materials and in comparison with alternative experimental methods such as elemental analysis. The CHNS-O comparison is made between the present data obtained with SEM-EDXS instrument and EA 3000 elemental analyzer used in previews studies conducted by the authors. Results show a difference of composition among packaging from different countries that can affect the treatment adopted for its valorization and the strategies of circular economy.

Slippery when wet: The free surface of the articular cartilage.

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.

Effect of Nanostructured Scaffold on Human Adipose-Derived Stem Cells: Outcome of In Vitro Experiments.

This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced; by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient.

The synovial surface of the articular cartilage.

The articular cartilage has been the subject of a huge amount of research carried out with a wide array of different techniques. Most of the existing morphological and ultrastructural data on the this tissue, however, were obtained either by light microscopy or by transmission electron microscopy. Both techniques rely on thin sections and neither allows a direct, face-on visualization of the free cartilage surface (synovial surface), which is the only portion subject to frictional as well as compressive forces. In the present research, high resolution visualization by scanning electron microscopy and by atomic force microscopy revealed that the collagen fibrils of the articular surface are exclusively represented by thin, uniform, parallel fibrils evocative of the heterotypic type IX-type II fibrils reported by other authors, immersed in an abundant matrix of glycoconjugates, in part regularly arranged in phase with the D-period of collagen. Electrophoresis of fluorophore-labeled saccharides confirmed that the superficial and the deeper layers are quite different in their glycoconjugate content as well, the deeper ones containing more sulfated, more acidic small proteoglycans bound to thicker, more heterogenous collagen fibrils. The differences found between the synovial surface and the deeper layers are consistent with the different mechanical stresses they must withstand.

The structure of cortical bone as revealed by the application of methods for the calcified matrix study.

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.