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Recently, it has been reported that heterologous biomaterials, where the collagen matrix is preserved, seem to facilitate blood clotting and the subsequent invasion of repairing and regenerative cells. This study aimed at evaluating the healing of human extraction sockets grafted with a collagenated cortico-cancellous porcine bone (mp3®, OsteoBiol®, Tecnoss®, Giaveno, Italy) by synchrotron radiation X-ray microtomography (microCT) and histology in order to have a 3D quantitative characterization over time. Ridge preservation with porcine bone and collagen membrane was performed on 21 patients using a flapless approach and a secondary soft tissue closure. At the time of implant placement, six bone samples were harvested, two in the 3-month group, two in the 6-month group, one in the 12-month group post-surgery, and one spontaneously healed control, and evaluated by microCT and histology. MicroCT revealed that in the grafted sites there were a greater number of trabeculae, although they appeared thinner than in the control sites; these trabeculae homogenously filled the defects, suggesting an improved strength of the socket. Histology showed that over time, the amount of biomaterial decreased and the newly formed bone increased, while less dense bone with wider marrow spaces was detected in the control sample, supporting synchrotron findings. The morphometric data converge to indicate the suitability of porcine bone for the preservation of post-extraction sockets. The use of mp3 is encouraged to preserve and heal sockets.

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.

This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block-DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells' morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.

The physico-chemical properties and in vivo efficacies of two nanoparticulate systems delivering the antiparkinsonian drug bromocriptine (BC) were compared in the present study. Monoolein Aqueous Dispersions (MADs) and Nanostructured Lipid Carriers (NLCs) were produced and characterized. Cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction revealed the morphology of MAD and NLC. Dimensional distribution was determined by Photon Correlation Spectroscopy (PCS) and Sedimentation Field Flow Fractionation (SdFFF). In particular, BC was shown to be encapsulated with high entrapment efficiency both in MAD and in NLC, according to SdFFF combined with HPLC. Two behavioral tests specific for akinesia (bar test) or akinesia/bradykinesia (drag test) were used to compare the effects of the different BC formulations on motor disabilities in 6-hydroxydopamine hemilesioned rats in vivo, a model of Parkinson's disease. Both free BC and BC-NLC reduced the immobility time in the bar test and enhanced the number of steps in the drag test, although the effects of encapsulated BC were longer lasting (5h). Conversely, BC-MAD was ineffective in the bar test and improved stepping activity in the drag test to a much lower degree than those achieved with the other preparations. We conclude that MAD and NLC can encapsulate BC, although only NLC provide long-lasting therapeutic effects possibly extending BC half-life in vivo.

Langmuir 32(3), 873 - 881(2016). doi:10.1021/acs.langmuir.5b03507, Structural properties and polymorphism of monoolein (MO) in aqueous solutions have been studied for a long time, and the final picture can be considered definite. The presence of bicontinuous phases and the ability to encapsulate hydrophilic, hydrophobic, and amphiphilic compounds, together with the capability to protect and slowly release the entrapped molecules, designated MO phases as good matrices for the sustained release of drugs. Because phase stability, loading efficiency, and bioavailability are strongly correlated, the interplay between MO phases and entrapped compounds is worthy of investigation. In this paper, low angle X-ray diffraction has been used to describe the effects of a model protein (the cytochrome-c) on the monoolein cubic phases as a function of both incubation time and protein concentration in the soaking solutions. Results show that the MO polymorphism is strongly modified by the protein, underlying the very large affinity of the cytochrome-c toward monoolein. However, the different phases have a different sensibility to cytochrome-c, as phase transitions occur when the protein amount exceeds some different critical values, probably related to the structure characteristics (2 cytochrome-c per unit cell at the Pn3m to Im3m cubic phase transition and 10–20 cytochrome-c per unit cell at the Im3m to P4$_3$32 cubic phase transition). Moreover, although equilibration times resulted to be quite long (more than 10 days), the fraction of cytochrome-c incorporated into the MO phases is very high (up to 20% v/v inside the P4$_3$32 cubic phase). Such results are intriguing: even if they may be specific to the cytochrome-c/MO case, the need of assessing the structural characteristics of lipid matrices before their use as drug delivery systems is evident., Published by ACS Publ., Washington, DC

Introduction Successful bone regeneration using both granules and blocks of biphasic calcium phosphate materials has been reported in the recent literature, in some clinical applications for maxillary sinus elevation, but the long-term kinetics of bone regeneration has still not been fully investigated. Materials and methods Twenty-four bilateral sinus augmentation procedures were performed and grafted with hydroxyapatite/β-tricalcium phosphate 30/70, 12 with granules and 12 with blocks. The samples were retrieved at different time points and were evaluated for bone regeneration, graft resorption, neovascularization, and morphometric parameters by computed microtomography and histology. Results A large amount of newly formed bone was detected in the retrieved specimens, together with a good rate of biomaterial resorption and the formation of a homogeneous and rich net of new vessels. The morphometric values were comparable at 5/6 months from grafting but, 9 months after grafting, revealed that the block-based specimens mimicked slightly better than granule-based samples the healthy native bone of the maxillary site. Conclusion The scaffold morphology was confirmed to influence the long-term kinetics of bone regeneration.

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.

Phase behaviour and structural properties of monoacylglycerides in water have been investigated for a long time, due to their extended polymorphism. In particular, monoolein (MO) in water shows several mesophases, characterized by a high disordered conformation of the hydrocarbon chains. At such conditions, an embedded protein can influence the physical properties of the lipid matrix, depending on the protein size and polarity. We take advantage of the structural properties of monoolein and cytochrome-c to extensively study the temperature effects on the cubic transition from Pn3m to Im3m by means of small-angle X-ray scattering technique (SAXS) and electronic absorption spectroscopy (EAS). To do so, we made samples composed of monoolein (50 mg/ml) in the presence of 1, 10 and 50 mg/ml of cytochrome-c. Ours preliminary SAXS results indicate that cyto-c is able to change the monoolein water channels, from cubic Pn3m to Im3m. Moreover, such kinetic behaviour is too slow, taking place within some days. EAS measurements indicate that the incorporation of cyto-c within the Monoolein water channels begins after two or three days (after the sample preparation). Besides, after one week of sample preparation the amount of cyto-c within the Monoolein water channels is equal to 60% and 34% for 1 and 10 mg/ml of cyto-c, respectively. Interestingly, increasing the temperature, the unity cell parameter decreases, indicating that water is going out from the unity cell and the symmetry of the liquid crystal phase can changes to another cubic or to hexagonal, depending on the temperature and the sample composition. We believe that these results could bring more insights on the protein-liquid crystal interaction.