Adriano Ferrari

Immunohistochemical study of muscle biopsy in children with cerebral palsy

Muscle biopsy was examined in 20 children with cerebral palsy, using immunohistochemical methods for marker of denervation neural cell adhesion molecules (N-CAM) in addition to standard techniques. Histological and histochemical study showed mild myopathic changes, type 1 predominance, and type 1 and type 2 hypotrophy, in accord with previous observations. Immunohistochemical study showed N-CAM expression in most biopsies (15/20), usually in scattered fibers, whereas in four patients aged less than 6 years it was expressed in grouped fibers. Our study supports the hypothesis of motor unit remodeling as a consequence of spasticity, especially in early phases of the disease.

Epidemiological survey of amyotrophic lateral sclerosis in the province of Reggio Emilia, Italy: influence of environmental exposure to lead.

We carried out a retrospective incidence, prevalence and mortality survey of amyotrophic lateral sclerosis (ALS) in the province of Reggio Emilia, northern Italy. Based on 79 patients, the mean incidence per year for the period 1980 through 1992 was 1.5 cases per 100,000. On December 31st, 1992, the prevalence rate was 5.4 per 100,000. In the 10-year period of 1983-1992 the average mortality rate was 1.3 per 100,000 per year. The average age at onset was 61.3 +/- 10.2, the average survival period thereafter was 26.3 months +/- 17.7; 27.3 +/- 17.6 for classic ALS, 19.5 +/- 8.4 for progressive bulbar palsy and 36.3 +/- 41.4 for pseudopolyneuritic ALS. The incidence rate, recorded in public health district No.12, an area with documented lead pollution since the 1970s, was standardized to the sex and age of the population of the province. Its incidence and prevalence rate were comparable to the rates found in the remaining area of the province.

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.

Human Dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations

BackgroundHuman dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency.ResultsThe STRO-1+/c-Kit+/CD34+ hDPSCs showed a slower proliferation, gradual loss of stemness, early cell senescence and apoptosis, compared to STRO-1+/c-Kit+/CD34− hDPSCs. Both the subpopulations demonstrated similar abilities to differentiate towards mesoderm lineages, whereas a significant difference was observed after the neurogenic induction, with a greater commitment of STRO-1+/c-Kit+/CD34+ hDPSCs. Moreover, undifferentiated STRO-1+/c-Kit+/CD34− hDPSCs did not show any expression of CD271 and nestin, typical neural markers, while STRO-1+/c-Kit+/CD34+ hDPSCs expressed both.ConclusionsThese results suggest that STRO-1+/c-Kit+/CD34− hDPSCs and STRO-1+/c-Kit+/CD34+ hDPSCs might represent two distinct stem cell populations, with different properties. These results trigger further analyses to deeply investigate the hypothesis that more than a single stem cell population resides within the dental pulp, to better define the flexibility of application of hDPSCs in regenerative medicine.

Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix.

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.

Differences and variations in the patterns of early independent walking

The early development of walking was investigated in 25 normal fullterm and in 25 low-risk preterm infants. All subjects were videorecorded within 3-4 weeks of the beginning of independent walking and again 4 months later. Analysis of the videos was carried out according to a predetermined list of items, with a semiquantitative score for each. The results indicate wide interindividual differences in normal infants in the form of independent walking. Several items seemed to be associated in different ways in different individuals. Age of onset, as long as the corrected age was considered, did not differ between preterm and fullterm infants, neither did their walking patterns. Gait asymmetries were often observed in both groups and they were related with asymmetries observable in prewalking locomotor behaviour. Toe-strike often occurred at the beginning of walking, but not after 4 months; in preterm infants the toe strike pattern correlated significantly with certain motor characteristics observed during the first weeks of life.

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.

Upper limb children action-observation training (UP-CAT): a randomised controlled trial in Hemiplegic Cerebral Palsy

BackgroundRehabilitation for children with hemiplegic cerebral palsy (HCP) aimed to improve function of the impaired upper limb (UL) uses a wide range of intervention programs. A new rehabilitative approach, called Action-Observation Therapy, based on the recent discovery of mirror neurons, has been used in adult stroke but not in children. The purpose of the present study is to design a randomised controlled trial (RCT) for evaluating the efficacy of Action-Observation Therapy in improving UL activity in children with HCP.Methods/DesignThe trial is designed according to CONSORT Statement. It is a randomised, evaluator-blinded, match-pair group trial. Children with HCP will be randomised within pairs to either experimental or control group. The experimental group will perform an Action-Observation Therapy, called UP-CAT (Upper Limb-Children Action-Observation Training) in which they will watch video sequences showing goal-directed actions, chosen according to children UL functional level, combined with motor training with their hemiplegic UL. The control group will perform the same tailored actions after watching computer games. A careful revision of psychometric properties of UL outcome measures for children with hemiplegia was performed. Assisting Hand Assessment was chosen as primary measure and, based on its calculation power, a sample size of 12 matched pairs was established. Moreover, Melbourne and ABILHAND-Kids were included as secondary measures. The time line of assessments will be T0 (in the week preceding the onset of the treatment), T1 and T2 (in the week after the end of the treatment and 8 weeks later, respectively). A further assessment will be performed at T3 (24 weeks after T1), to evaluate the retention of effects. In a subgroup of children enrolled in both groups functional Magnetic Resonance Imaging, exploring the mirror system and sensory-motor function, will be performed at T0, T1 and T2.DiscussionThe paper aims to describe the methodology of a RCT for evaluating the efficacy of Action-Observation Therapy in improving UL activity in children with hemiplegia. This study will be the first to test this new type of treatment in childhood. The paper presents the theoretical background, study hypotheses, outcome measures and trial methodology.Trial RegistrationNCT01016496

Randomized Trial of Observation and Execution of Upper Extremity Actions Versus Action Alone in Children With Unilateral Cerebral Palsy

Background. The properties of the mirror neuron system suggest a new type of upper limb (UL) rehabilitation in children with unilateral cerebral palsy (UCP), based on observation of action therapy followed by execution of a variety of observed movements (AOT). Objective. We tested the effects of AOT in the Upper Limb Children Action Observation Training (UP-CAT) trial. Methods. In a randomized, evaluator-blinded, block-designed trial, 24 UCP children with mild to moderate hand impairment were assigned to 2 groups. The experimental group observed, 1 hour per day for 3 consecutive weeks, video sequences of unimanual or bimanual goal-directed actions and subsequently executed observed actions with the hemiparetic UL or both ULs. The control group performed the same actions in the same order as the experimental sample, but had watched computer games. The Assisting Hand Assessment (AHA) scale was the primary outcome measure; the Melbourne assessment and ABILHAND-Kids were secondary ones. Outcomes were assessed at 1 week (T1), 8 weeks (T2), and 24 weeks (T3) after the end of the training. Results. The experimental group improved more (P = .008) in score changes for the AHA at the primary endpoints T1 (P = .008), T2 (P = .019), and T3 (P = .049). No between-group significant changes were found for ABILHAND-Kids or Melbourne assessment. Conclusions. UP-CAT improved daily UL activities in UCP children, suggesting a new rehabilitation approach based on a neurophysiological model of motor learning.