Franco Rustichelli

Three Years After Transplants in Human Mandibles, Histological and In-Line Holotomography Revealed That Stem Cells Regenerated a Compact Rather Than a Spongy Bone: Biological and Clinical Implications

Mesenchymal stem cells deriving from dental pulp differentiate into osteoblasts capable of producing bone. In previous studies, we extensively demonstrated that, when seeded on collagen I scaffolds, these cells can be conveniently used for the repair of human mandible defects. Here, we assess the stability and quality of the regenerated bone and vessel network 3 years after the grafting intervention, with conventional procedures and in‐line holotomography, an advanced phase‐imaging method using synchrotron radiation that offers improved sensitivity toward low‐absorbing structures. We found that the regenerated tissue from the graft sites was composed of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Thus, the regenerated bone, being entirely compact, is completely different from normal alveolar bone. Although the bone regenerated at the graft sites is not of the proper type found in the mandible, it does seem to have a positive clinical impact. In fact, it creates steadier mandibles, may well increase implant stability, and, additionally, may improve resistance to mechanical, physical, chemical, and pharmacological agents.

Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone

ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.

Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.

Micromechanics of bone tissue-engineering scaffolds, based on resolution error-cleared computer tomography.

Synchrotron radiation micro-computed tomography (SRmuCT) revealed the microstructure of a CEL2 glass-ceramic scaffold with macropores of several hundred microns characteristic length, in terms of the voxel-by-voxel 3D distribution of the attenuation coefficients throughout the scanned space. The probability density function of all attenuation coefficients related to the macroporous space inside the scaffold gives access to the tomograph-specific machine error included in the SRmuCT measurements (also referred to as instrumental resolution function). After Lorentz function-based clearing of the measured CT data from the systematic resolution error, the voxel-specific attenuation information of the voxels representing the solid skeleton is translated into the composition of the material inside one voxel, in terms of the nanoporosity embedded in a dense CEL2 glass-ceramic matrix. Based on voxel-invariant elastic properties of dense CEL2 glass-ceramic, continuum micromechanics allows for translation of the voxel-specific nanoporosity into voxel-specific elastic properties. They serve as input for Finite Element analyses of the scaffold structure. Youngs modulus of a specific CT-scanned macroporous scaffold sample, predicted from a Finite Element simulation of a uniaxial compression test, agrees well with the experimental value obtained from an ultrasonic test on the same sample. This highlights the satisfactory predictive capabilities of the presented approach.

Stem cell tracking by nanotechnologies

Advances in stem cell research have provided important understanding of the cell biology and offered great promise for developing new strategies for tissue regeneration. The beneficial effects of stem cell therapy depend also by the development of new approachs for the track of stem cells in living subjects over time after transplantation. Recent developments in the use of nanotechnologies have contributed to advance of the high-resolution in vivo imaging methods, including positron emission tomography (PET), single-photon emission tomography (SPECT), magnetic resonance (MR) imaging, and X-Ray computed microtomography (microCT). This review examines the use of nanotechnologies for stem cell tracking.

Bone Turnover in Wild Type and Pleiotrophin-Transgenic Mice Housed for Three Months in the International Space Station (ISS)

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity’s negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

Osteointegration of bioactive glass-coated zirconia in healthy bone: an in vivo evaluation

Osteointegration of yttria stabilised tetragonal zirconia (YSTZ), either coated with bioactive glass named RKKP bioglaze (RKKP) or uncoated, was evaluated in an animal model. RKKP-coated and uncoated (controls) YSTZ cylinders were implanted in the distal femoral epiphyses of 14 Sprague Dawley rats under general anaesthesia. At the experimental times of 30 and 60 days after sacrifice, histomorphometry and SEM microanalysis were performed on methylmethacrylate-embedded undecalcified sections to determine the osteointegration rate. At 30 days, a significantly higher affinity index was demonstrated in vivo by histomorphometric evaluation in RKKP-coated versus uncoated YSTZ implants p < 0.05); at 60 days, the coated implants behaved better than controls (affinity index of + 32%), but the difference observed lay within the statistical uncertainty. SEM analysis demonstrated better bone adhesion to the material in RKKP-coated YSTZ at both 30 and 60 days. These findings suggest that YSTZ coated with the bioactive glass named RKKP enhances osteointegration of ceramics.

X-Ray Synchrotron Radiation Pseudo-Holotomography as a New Imaging Technique to Investigate Angio- and Microvasculogenesis with No Usage of Contrast Agents

Standard X-ray micro-computed tomography is a technique that allows a good visualization of the structure of mineralized tissues and biomaterials, but it fails to finely discern soft tissues. Here, we used X-ray synchrotron radiation pseudo-holotomography to visualize, at three-dimensional (3D) level, microvascular networks for the first time with no need for contrast agents, and to extract quantitative structural data in a bone-engineered construct implanted for 24 weeks in a mouse. When compared to standard histology, pseudo-holotomography allowed a previously unavailable 3D resolution of the vessels, which in turn appeared more clearly visible. Thus, pseudo-holotomography is an innovative technique that offers a promising powerful tool to investigate angio- and microvasculogenesis in advanced biomedical research areas such as regenerative medicine and antiangiogenic cancer therapies.

High-resolution X-ray microtomography for three-dimensional visualization of human stem cell muscle homing

In the perspective of clinical translation of stem cell research, it would be advantageous to develop new techniques to detect donor cells after transplantation to track their fate and thus better understand their role in regeneration of damaged and diseased tissues. In this study we use X‐ray computed microtomography for three‐dimensional visualization of stem cells that were labeled with magnetic nanoparticles and transplanted via intra‐arterial infusion. We show that X‐ray computed microtomography offers the possibility to detect with high definition and resolution human cells after transplantation, and opens new possibilities for both experimental stem cell research.

Neutron tomography for archaeological investigations

Within the last decade neutron tomography and radiography significantly gained importance. Especially its application in non-destructive testing for industrial components can be underlined. A good example is the automotive and aviation industry, where a high contrast for the used lubricants and adhesive materials is required. In contrast to X-rays, neutrons are able to penetrate thick layers of metals and provide on the other hand a high sensitivity to hydrogen containing materials. In recent years, a large number of applications in other fields like biology, medicine, geology and especially archaeology have been reported. Here the potential of neutron tomography for investigations on archaeological samples shall be outlined and some recent examples will be presented.