Davide Liccardo

Three-Dimensional imaging of the developing vasculature within stem cell-seeded scaffolds cultured in ovo

Successful tissue engineering requires functional vascularization of the three-dimensional constructs with the aim to serve as implants for tissue replacement and regeneration. The survival of the implant is only possible if the supply of oxygen and nutrients by developing capillaries from the host is established. The chorioallantoic membrane (CAM) assay is a valuable tool to study the ingrowth and distribution of vessels into scaffolds composed by appropriate biomaterials and stem cell populations that are used in cell-based regenerative approaches. The developing vasculature of chicken embryos within cell-seeded scaffolds can be visualized with microcomputed tomography after intravenous injection of MicroFil®, which is a radiopaque contrast agent. Here, we provide a step-by-step protocol for the seeding of stem cells into silk fibroin scaffolds, the CAM culture conditions, the procedure of MicroFil® perfusion, and finally the microcomputed tomography scanning. Three-dimensional imaging of the vascularized tissue engineered constructs provides an important analytical tool for studying the potential of cell seeded scaffolds to attract vessels and form vascular networks, as well as for analyzing the number, density, length, branching, and diameter of vessels. This in ovo method can greatly help to screen implants that will be used for tissue regeneration purposes before their in vivo testing, thereby reducing the amount of animals needed for pre-clinical studies.

NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact.

Synchrotron Phase Tomography: An Emerging Imaging Method for Microvessel Detection in Engineered Bone of Craniofacial Districts

The engineering of large 3D constructs, such as certain craniofacial bone districts, is nowadays a critical challenge. Indeed, the amount of oxygen needed for cell survival is able to reach a maximum diffusion distance of ~150–200 μm from the original vascularization vector, often hampering the long-term survival of the regenerated tissues. Thus, the rapid growth of new blood vessels, delivering oxygen and nutrients also to the inner cells of the bone grafts, is mandatory for their long-term function in clinical practice. Unfortunately, significant progress in this direction is currently hindered by a lack of methods with which to visualize these processes in 3D and reliably quantify them. In this regard, a challenging method for simultaneous 3D imaging and analysis of microvascularization and bone microstructure has emerged in recent years: it is based on the use of synchrotron phase tomography. This technique is able to simultaneously identify multiple tissue features in a craniofacial bone site (e.g., the microvascular and the calcified tissue structure). Moreover, it overcomes the intrinsic limitations of both histology, achieving only a 2D characterization, and conventional tomographic approaches, poorly resolving the vascularization net in the case of an incomplete filling of the newly formed microvessels by contrast agents. Indeed, phase tomography, being based on phase differences among the scattered X-ray waves, is capable of discriminating tissues with similar absorption coefficients (like vessels and woven bone) in defined experimental conditions. The approach reviewed here is based on the most recent experiences applied to bone regeneration in the craniofacial region.

A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo

Pentose phosphate pathway (PPP) is a major glucose metabolism pathway, which has a fundamental role in cancer growth and metastasis. Even though PPP blockade has been pointed out as a very promising strategy against cancer, effective anti-PPP agents are not still available in the clinical setting. Here we demonstrate that the natural molecule polydatin inhibits glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP. Polydatin blocks G6PD causing accumulation of reactive oxygen species and strong increase of endoplasmic reticulum stress. These effects are followed by cell cycle block in S phase, an about 50% of apoptosis, and 60% inhibition of invasion in vitro. Accordingly, in an orthotopic metastatic model of tongue cancer, 100 mg/kg polydatin induced an about 30% tumor size reduction with an about 80% inhibition of lymph node metastases and 50% reduction of lymph node size (p < 0.005). Polydatin is not toxic in animals up to a dose of 200 mg/kg and a phase II clinical trial shows that it is also well tolerated in humans (40 mg twice a day for 90 days). Thus, polydatin may be used as a reliable tool to limit human cancer growth and metastatic spread.

Evaluation and Isolation of Cancer Stem Cells Using ALDH Activity Assay

The aldehyde dehydrogenase (ALDH) is a polymorphic enzyme responsible for the oxidation of aldehydes to carboxylic acids. In this chapter, it is described the role of ALDH in the identification of cancer stem cells (CSCs), having been shown that stem cells express high levels of ALDH. Here, we present a method called ALDEFLUOR assay used for the identification, evaluation, and isolation of normal, cancer stem and progenitor cells.

Pentose phosphate pathway inhibition induce Endoplasmic Reticulum stress and autophagy

Pentose phosphate pathway (PPP) is a major glucose catabolism pathway that supplies the cell with a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. NADPH is necessary for the detoxification of reactive oxygen species (ROS) and reductive biosynthesis. A key player in this pathway is the enzyme glucose-6-phosphate dehydrogenase (G6PD) that reduces NADP+ to NADPH, oxidizes glucose-6-phosphate and prevents ROS accumulation. Here, we show that the natural molecule 3,4’,5-trihydroxystilbene-3-β-d-glucoside (Polydatin) inhibits glucose-6-phosphate dehydrogenase (G6PD). As expected, G6PD inhibition causes an imbalance in NADP+/NADPH ratio, leading to a redox imbalance, followed by Endoplasmic Reticulum (ER) stress, autophagy, cell cycle block and apoptosis. we have demonstrated a link between G6PD inhibition and ER stress, showing that Unfolded Protein Response mediator such as PERK and IRE-1 have a key role in inducing autophagy and apoptosis after PPP block. Moreover, combination of PPP inhibition with autophagy inhibitors, such as chloroquine, strongly potentiate cytotoxicity on cancer cells, evidencing the role of autophagy as an escaping mechanism. This results shows that double inhibition of PPP and autophagy may be an affective therapeutic strategy against cancer.