Gian Maria Cavallini

Human Serum Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro and In Vivo

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

Bimanual microphacoemulsification versus coaxial miniphacoemulsification : Prospective study

PURPOSE: To compare the outcomes of bimanual microphacoemulsification and coaxial miniphacoemulsification and assess the potential advantages of the former over the latter. SETTING: Institute of Ophthalmology, University of Modena, Modena, Italy. METHODS: In a controlled prospective clinical trial, 100 eyes of 50 patients with nuclear or corticonuclear cataract of grade 2 to 4 on the Lens Opacities Classification System III had phacoemulsification. Fifty eyes were randomized to have surgery by the bimanual technique and 50, by the coaxial technique. All surgeries were performed by the same surgeon using the same machine (Sovereign WhiteStar, American Medical Optics). In all cases, the incision was made superiorly in clear cornea and a hydrophobic acrylic flexible intraocular lens (Acri.Smart 48 S, Acri.Tec) was implanted. Intraoperative parameters were mean phacoemulsification time, total phacoemulsification percentage, effective phacoemulsification time (EPT), total volume of balanced salt solution (BSS) used, total surgical time, and final size of the corneal incision. Postoperative parameters were visual acuity, astigmatism changes by vector analysis, corneal thickness, endothelial cell count, and presence of flare and cells in the anterior chamber. RESULTS: The only statistically significant difference between the 2 groups was the total volume of the BSS used (P = .004) and total surgical time (P = .045). CONCLUSIONS: Both techniques were safe and effective for cataract surgery. With bimanual microphacoemulsification, significantly less BSS was used and the total surgical time was significantly shorter than with the coaxial method.

Normal intraocular pressure in children

The age-related trend values and the normal intraocular pressure (IOP) increase curve from birth through the 16th year of life were studied in 460 subjects with a noncontact tonometer (Keeler Pulsair, Keeler, Ltd, Windsor, Berks, UK). Much lower values than in adults were recorded in subjects up to the age of 3 or 4 years. This finding leads us to believe that in the treatment of infantile glaucoma IOP should be kept within the age physiologic levels, in an attempt to prevent visual field loss and optic atrophy.

In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp

AIM To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

Melanocortins protect against progression of Alzheimer's disease in triple- transgenic mice by targeting multiple pathophysiological pathways

Besides specific triggering causes, Alzheimers disease (AD) involves pathophysiological pathways that are common to acute and chronic neurodegenerative disorders. Melanocortins induce neuroprotection in experimental acute neurodegenerative conditions, and low melanocortin levels have been found in occasional studies performed in AD-type dementia patients. Here we investigated the possible neuroprotective role of melanocortins in a chronic neurodegenerative disorder, AD, by using 12-week-old (at the start of the study) triple-transgenic (3xTg-AD) mice harboring human transgenes APPSwe, PS1M146V, and tauP301L. Treatment of 3xTg-AD mice, once daily until the end of the study (30 weeks of age), with the melanocortin analog [Nle(4),D-Phe(7)]-α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus phosphorylation/level of all AD-related biomarkers investigated (mediators of amyloid/tau cascade, oxidative/nitrosative stress, inflammation, apoptosis), decreased neuronal loss, induced over-expression of the synaptic activity-dependent gene Zif268, and improved cognitive functions, relative to saline-treated 3xTg-AD mice. Pharmacological blockade of melanocortin MC4 receptors prevented all neuroprotective effects of NDP-α-MSH. Our study identifies, for the first time, a class of drugs, MC4 receptor-stimulating melanocortins, that are able to counteract the progression of experimental AD by targeting pathophysiological mechanisms up- and down-stream of β-amyloid and tau. These data could have important clinical implications.

The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes.

Background:The association between HAART and lipodystrophy is well established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. Methods:SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10–200 &mgr;mol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2-) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2- were studied by flow cytometry and multispectral imaging flow cytometry. Results:In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. Conclusion:Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.

Quantitative Clinical Anatomy of the Human Cornea in vivo

The cornea is the most important refractive element in the human ocular system, providing approximately two thirds of the eyes refractive power in the nonaccommodative state. The methods of description and analysis of the corneal anatomy in vivo continue to be refined thanks to the increased interest aroused by the advent of corneal refractive surgery, which aims to modify the dioptric power of the ocular system by altering the thickness and the radius of curvature of the cornea. In the present study we report quantitative morphometric data of corneal thickness and the radius of curvature of the anterior surface of the cornea obtained in vivo from normal human subjects using ultrasonic pachymetry and computer-assisted topographic videokeratoscopy. We found a highly significant statistical correlation in the distribution of corneal thickness and the radius of curvature of the anterior surface of the cornea along the principal corneal meridians (horizontal and vertical meridians) and within three different concentric zones of the cornea, 2, 4 and 6 mm, respectively, from the geometric center along the principal meridians. Correlation was also found for the same morphometric parameters between the right and the left eye. By contrast, no correlation was found between corneal thickness and the radius of curvature of the anterior surface of the cornea. These findings suggest that the adult human cornea is a structure characterized by a highly ordered tridimensional architecture and symmetry and that a specific distribution of corneal thickness and the radius of curvature of the anterior corneal surface along the principal meridians may be essential for the refractive function of the human corneal diopter in vivo.

Visual recovery after scleral buckling for macula-off retinal detachments: An optical coherence tomography study

Purpose To assess the postoperative macular reattachment through OCT3 in eyes treated with episcleral surgery due to macula-off rhegmatogenous retinal detachment, as well as to verify if there is a statistically relevant relation between the persistence of a subfoveal detachment and poor postoperative functional recovery. Methods Twelve eyes of 12 patients who underwent episcleral surgery due to macula-off rhegmatogenous retinal detachment were enrolled and examined in a prospective study. Exclusion criteria were the following: traumatic retinal detachments, detachment relapses, macular holes, amblyopia, and grade B proliferative vitreoretinopathy or higher. The time period from the onset of subjective symptoms of retinal detachment to retinal surgery ranged from 3 to 7 days. All patients were evaluated in the preoperative and the postoperative period (after 1, 3, and 6 months) through measurement of visual acuity by ETDRS charts, fundus photographs, and macular tomography with OCT3. The postoperative tomography outcomes and the visual acuity were statistically examined using the Mann-Whitney U-test. Results One month after surgery, despite the macular reattachment assessable ophthalmoscopically and through fundus photographs, the OCT examination showed macular subretinal fluid persistence in 66.6% of cases. After 3 and 6 months, the persistence of such foveal detachment was respectively observed in 41.6% and in 33.3% of cases. Moreover, the macular subretinal fluid persistence in the postoperative period showed a statistically significant relation with poor functional recovery. Conclusions Delayed or incomplete visual recovery after episcleral surgery for macula-off retinal detachment may be related to macular subretinal fluid persistence, assessable with tomography and not visible ophthalmoscopically.

Impact of preoperative testing on ophthalmologic and systemic outcomes in cataract surgery

Purpose To evaluate the incidence of ophthalmologic and systemic complications in patients who undergo cataract surgery without preoperative tests compared to subjects undergoing cataract surgery preceded by preoperative tests. Methods The randomized controlled study included 1276 consecutive patients admitted to the Institute of Ophthalmology of the University of Modena and Reggio Emilia for cataract surgery. The patients were randomly divided into two groups: 638 were assigned not to undergo preoperative evaluation based on routine medical tests and electrocardiograms; the other 638 underwent preoperative evaluation based on said tests. Ophthalmologic and systemic complications were assessed intraoperatively and 1 month after surgery. Results Eleven intraoperative complications occurred in the group without preoperative tests and eight in the group with preoperative tests; at 1 month six complications were recorded in the group without tests and five in the group with tests. Systemic adverse events occurred intraoperatively in four patients, whereas no systemic adverse event was recorded at 1 month in either group. No statistically significant differences were observed between the two groups. Conclusions The findings of this study have broad applicability, because the sample is representative of the population existing in numerous social and healthcare settings; they are of value for administrative purposes, because they may be taken as reference in resource allocation plans; and they have medicolegal implications, as the resulting conduct of healthcare providers is supported by a rigorous scientific study.

From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU

In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell-based corneal therapy.