Alessandra Giuliani

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.

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.

Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone.

Lipocalin‐2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over‐expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed‐down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate‐resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor‐κB ligand (RANKL) and of the IL‐6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment. J. Cell. Physiol. 228: 2210–2221, 2013.

In Vivo Regenerative Properties of Coralline‐Derived (Biocoral) Scaffold Grafts in Human Maxillary Defects: Demonstrative and Comparative Study with Beta‐Tricalcium Phosphate and Biphasic Calcium Phosphate by Synchrotron Radiation X‐Ray Microtomography

BACKGROUND In recent years, there has been interest on the fabrication of systems using particulates or block-based approach for bone tissue engineering (TE) scaffolds, possessing porous interconnected structures. In fact, these particular morphologies greatly increase the surface area for more chemical and biological reactions to take place. PURPOSE This study was designed to demonstrate the unique capability of the synchrotron radiation x-ray microtomography (micro-CT) in offering an advanced characterization of coralline-derived (Biocoral) biomaterials placed in human maxillary defects as it allows, in a nondestructive way, a complete, precise, and high-resolution three-dimensional analysis of their microstructural parameters. Moreover, the comparison between Biocoral and other biomaterials was explored to understand the mechanism of their biological behavior as bone substitute. MATERIALS AND METHODS Implant survival, bone regeneration, graft resorption, neovascularization, and morphometric parameters (including anisotropy and connectivity index of the structures) were evaluated by micro-CT in Biocoral and the other biomaterials after 6 to 7 months from implantation in human maxillary bone defects. RESULTS After the in vivo tests, a huge amount of bone was detected in the retrieved Biocoral-based samples, coupled with a good rate of biomaterial resorption and the formation of a homogeneous and rich net of new vessels. The morphometric parameters were comparable to those obtained in the biphasic calcium phosphate-based control, with the exception of the connectivity index for which this control exhibited the most well-connected structure. This last result, together with those referred to the poor performances of the β-tricalcium phosphate block-based sample, suggests that the particular scaffold morphology may play a role in the hunt the optimal scaffold structure to be implanted. CONCLUSION In this limited study, implant success rate seems not strictly dependent on the biomaterial that is used, but on the scaffold morphology. Micro-CT technique was demonstrated to play a fundamental role in advanced characterization of bone TE constructs.

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal–vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration.

Osteogenic potential of dualblocks cultured with human periodontal ligament stem cells: in vitro and synchrotron microtomography study

BACKGROUND AND OBJECTIVE In the present study, the early stages of in vitro bone formation in collagenated porcine scaffolds cultured with human periodontal ligament cells were investigated. The comparison between the osteogenic potential of this structure in basal and differentiating culture media was explored to predict the mechanism of its biological behavior as graft in human defect. Results were validated by synchrotron radiation X-Ray phase contrast computed microtomography (micro-CT). As the periodontal disease plays a key role in systemic and oral diseases, it is crucial to find advanced therapeutic clinical interventions to repair periodontal defects. This has been recently explored using cells and tissues developed in vitro that should ideally be immunologically, functionally, structurally and mechanically identical to the native tissue. MATERIAL AND METHODS In vitro cultures of human periodontal ligament cells, easily obtained by scraping of alveolar crestal and horizontal fibers of the periodontal ligament, were seeded on to collagenated porcine blocks constituted by natural cancellous and cortical bone. 3D images were obtained by synchrotron radiation micro-CT and processed with a phase-retrieval algorithm based on the transport of intensity equation. RESULTS Starting from the second week of culture, newly formed mineralized bone was detected in all the scaffolds, both in basal and differentiating media. Bone mineralization was proved to occur preferentially in the trabecular portion and in differentiating media. CONCLUSION The chosen method, supported by phase contrast micro-CT analysis, successfully and quantitatively monitored the early stages of bone formation and the rate of the bioscaffold resorption in basal and differentiating culture media.

High-resolution X-ray microtomography for three-dimensional imaging of cardiac progenitor cell homing in infarcted rat hearts

The recent introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathological heart and has opened a search for new therapeutic strategies. Recent published reports have contributed to identifying possible cellular therapy approaches to generate new myocardium, involving transcoronary and intramyocardial injection of progenitor cells. However, one of the limiting factors in the overall interpretation of clinical results obtained by cell therapy is represented by the lack of three‐dimensional (3D) high‐resolution methods for the visualization of the injected cells and their fate within the myocardium. This work shows that X‐ray computed microtomography may offer the unique possibility of detecting, with high definition and resolution and in ex vivo conditions, the 3D spatial distribution of rat cardiac progenitor cells, labelled with iron oxide nanoparticles, inside the infarcted rat heart early after injection. The obtained 3D images represent a very innovative progress as compared to experimental two‐dimensional (2D) histological analysis, which requires time‐consuming energies for image reconstruction in order to provide the overall distribution of rat clonogenic cells within the heart. Through microtomography, we were able to observe in 3D the presence of these cells within damaged cardiac tissue, with important structural details that are difficult to visualize by conventional bidimensional imaging techniques. This new 3D‐imaging approach appears to be an important way to investigate the cellular events involved in cardiac regeneration and represents a promising tool for future clinical applications. Copyright

Purified collagen I oriented membrane for tendon repair: an ex vivo morphological study.

Injured tendons have limited repair ability after full‐thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. The multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham‐operated controls). Animals were euthanized 1 or 6 months after surgery, and tendons were subjected to histological and Synchrotron Radiation‐based Computed Microtomography (SRµCT) examination and 3D structure analysis. Histological and SRµCT findings showed satisfactory graft integration with native tendon. Histological examination also showed ongoing angiogenesis. Adverse side‐effects (inflammation, rejection, calcification) were not observed. The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation.

Organization of Extracellular Matrix Fibers Within Polyglycolic Acid–Polylactic Acid Scaffolds Analyzed Using X-Ray Synchrotron-Radiation Phase-Contrast Micro Computed Tomography

Spatiotemporal organized patterns of cell surface-associated and extracellular matrix (ECM)-embedded molecules play important roles in the development and functioning of tissues. ECM proteins interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation., Using X-ray phase-contrast micro computed tomography (microCT), we visualized the three-dimensional (3D) image of ECM organization after in vitro seeding of bone marrow-derived human and murine mesenchymal stem cells (MSCs) induced to myogenic differentiation, labelled with iron oxide nanoparticles, and seeded onto polyglycolic acid-polylactic acid scaffolds. X-ray microCT enabled us to detect with high spatial resolution the 3D structural organization of ECM within the bioscaffold and how the presence of cells modified the construct arrangement. Species-specific differences between the matrix produced by human and murine cells were observed. In conclusion, X-ray synchrotron radiation microCT analysis appeared to be a useful tool to identify the spatiotemporal pattern of organization of ECM fibers within a bioscaffold.

Zirconia enriched dental adhesive: A solution for OCT contrast enhancement. Demonstrative study by synchrotron radiation microtomography

OBJECTIVE The major aim of this study was to prove the capability of the optical coherence tomography (OCT) method in visualizing the integrity of the adhesive fillings and of the interfaces between the adhesive, tooth structures and composite resin. As zirconium dioxide was added to the composition of the adhesive layer in order to strengthen the backscattered light in the OCT investigation, for a better visualization of the interfaces, the determination of a proper zirconia concentration was another aim of our study. METHOD Several class II cavities were prepared in human premolars and were filled with dental adhesive containing different zirconia concentrations and light-curing composite resin. Both OCT and synchrotron radiation microtomography (micro-CT) were used to analyse the morphology of the tooth-adhesive-composite interfaces and to investigate the adhesive layer. RESULTS The pore distribution, both at the interfaces level and in the resin, and the analysis of the adhesive layer integrity were obtained. A good agreement between OCT and micro-CT analyses was observed in terms of detecting discontinuities in the adhesive layer. Furthermore, micro-CT showed that zirconia percentages in the adhesive higher than 20 vol.% lead to conglomerates formation, which can negatively influence mechanical properties. Meanwhile, OCT confirmed a factor of 3 for the contrast enhancement when 20% of zirconia was included in the adhesive composition. SIGNIFICANCE The present study proved the capability of the OCT method in visualizing the morphology and integrity of zirconia doped tooth adhesive fillings, to be used for a further in vivo tool development.