Francesca Grandi

Outcome Comparison Among Laparoscopic Duhamel, Laparotomic Duhamel, and Transanal Endorectal Pull-Through: A Single-Center, 18-Year Experience

PURPOSE Transanal endorectal pull-through has changed the treatment of Hirschsprungs disease (HD) in the past decade. The aim of the study was to compare outcomes, obtained in a single center, with laparotomic Duhamel (LTD), laparoscopic Duhamel (LSD), and laparoscopic-assisted transanal endorectal pull-through (LTEPT). MATERIALS AND METHODS We retrospectively reviewed the charts of all patients operated on for HD since 1992. Preoperative, operative, and postoperative data were collected to compare short- and long-term outcomes among the three groups. RESULTS From 1992 to 2010, 70 children were treated for HD. Patients were divided into three groups based on the surgical technique used: 14 LTEPT, 32 LSD, and 24 LTD. Mean ages at surgery were 4.67, 14.61, and 13.28 months, respectively. Patients in the LTEPT group had significant shorter operating times (195 versus 257 versus 291 minutes, P=.03), earlier start of feeding (1.2 versus 3.1 versus 4.7 days, P<.01), and shorter length of hospital stay (4.4 versus 6.8 versus 9.7 days, P<.011). Overall complications rate was lower in the LTEPT (14%) than in the LSD (31.2%) and LTD (29.7%) groups. Postoperative enterocolitis incidence was 3%-4% in the Duhamel groups and none in LTEPT. Long-term outcome showed less constipation and better continence for age in the LTEPT group at the 1-year follow-up (P=.033). CONCLUSIONS This study further supports technical advantages, lighter impact of the surgical procedure on infants, lower incidence of complications, and better long-term outcome of the transanal pull-through compared to the Duhamel approaches.

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue‐engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein‐loading ability and biodegradability. Copyright

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

Pediatric Chronic Intestinal Failure in Italy: Report from the 2016 Survey on Behalf of Italian Society for Gastroenterology, Hepatology and Nutrition (SIGENP)

Background: Intestinal failure (IF) is the reduction in functioning gut mass below the minimal level necessary for adequate digestion and absorption of nutrients and fluids for weight maintenance in adults or for growth in children. There is a paucity of epidemiologic data on pediatric IF. The purpose of this study was to determine the prevalence, incidence, regional distribution and underlying diagnosis of pediatric chronic IF (CIF) requiring home parenteral nutrition (HPN) in Italy. Methods: Local investigators were selected in 19 Italian centers either of reference for pediatric HPN or having pediatric gastroenterologists or surgeons on staff and already collaborating with the Italian Society for Pediatric Gastroenterology, Hepatology and Nutrition with regard to IF. Data requested in this survey for children at home on Parenteral Nutrition (PN) on 1 December 2016 included patient initials, year of birth, gender, family’s place of residence and underlying diagnosis determining IF. Results: We recorded 145 CIF patients on HPN aged ≤19 years. The overall prevalence was 14.12/million inhabitants (95% CI: 9.20–18.93); the overall incidence was 1.41/million inhabitant years (95% CI: 0.53–2.20). Conclusion: Our survey provides new epidemiological data on pediatric CIF in Italy; these data may be quantitatively useful in developing IF care strategy plans in all developed countries.

Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton’s jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44+/CD49c+/CD49e+/CD151+/CD73+/CD49f−/CD26− resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

Partially oxidized polyvinyl alcohol conduitfor peripheral nerve regeneration

Surgical reconstruction of peripheral nerves injuries with wide substance-loss is still a challenge. Many studies focused on the development of artificial nerve conduits made of synthetic or biological materials but the ideal device has not yet been identified. Here, we manufactured a conduit for peripheral nerve regeneration using a novel biodegradable hydrogel we patented that is oxidized polyvinyl alcohol (OxPVA). Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) conduits, through in vitro and in vivo analysis. Unlike SF, OxPVA and neat PVA scaffolds did not support SH-SY5Y adhesion and proliferation in vitro. After implantation in rat model of sciatic nerve transection, the three conduits sustained the regeneration of the injured nerve filling a gap of 5 mm in 12 weeks. Implanted animals showed a good gait recovery. Morphometric data related to the central portion of the explanted conduit interestingly highlighted a significantly better outcome for OxPVA scaffolds compared to PVA conduits in terms of axon density, also with respect to the autograft group. This study suggests the potential of our novel biomaterial for the development of conduits for clinical use in case of peripheral nerve lesions with substance loss.

Biofabrication of a novel leukocyte-fibrin-platelet membrane as a cells and growth factors delivery platform for tissue engineering applications

Autologous platelet‐rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte‐fibrin‐platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin‐10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface‐protein level (CD44high/CD73+/CD34+/CD117+/CD31+). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.

Composite Scaffolds Based on Intestinal Extracellular Matrices and Oxidized Polyvinyl Alcohol: A Preliminary Study for a New Regenerative Approach in Short Bowel Syndrome

Pediatric Short Bowel Syndrome is a rare malabsorption disease occurring because of massive surgical resections of the small intestine. To date, the issues related to current strategies including intestinal transplantation prompted the attention towards tissue engineering (TE). This work aimed to develop and compare two composite scaffolds for intestinal TE consisting of a novel hydrogel, that is, oxidized polyvinyl alcohol (OxPVA), cross-linked with decellularized intestinal wall as a whole (wW/OxPVA) or homogenized (hW/OxPVA). A characterization of the supports was performed by histology and Scanning Electron Microscopy and their interaction with adipose mesenchymal stem cells occurred by MTT assay. Finally, the scaffolds were implanted in the omentum of Sprague Dawley rats for 4 weeks prior to being processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). In vitro studies proved the effectiveness of the decellularization, highlighting the features of the matrices; moreover, both supports promoted cell adhesion/proliferation even if the wW/OxPVA ones were more effective (p < 0.01). Analysis of explants showed a continuous and relatively organized tissue wall around the supports with a connective appearance, such as myofibroblastic features, smooth muscle, and epithelial cells. Both scaffolds, albeit with some difference, were promising; nevertheless, further analysis will be necessary.

Short Bowel Syndrome and Tissue Engineering: a preliminary study towards the development of a new regenerative approach in paediatric patients

Pediatric Short Bowel Syndrome (SBS) is a malabsorption state following massive surgical resections of the small intestine. The current therapeutic options issues (i.e. parental nutrition, surgical lengthening, transplantation) have prompt the research in Tissue Engineering. Thus, our aim was to preliminary investigate in vitro/in vivo two composite scaffolds for engineering the small intestine without resorting to autologous intestinal epithelial organoid units which, to date, are the cell source mainly considered for this purpose. In particular, we developed composite supports consisting of a novel biocompatible/resorbable cryogel that is oxidized polyvinyl alcohol (OxPVA) [1] crosslinked with intestinal mucosa whole (wIM/OxPVA) or homogenized (hIM/OxPVA). After evaluating the scaffolds by histology and Scanning Electron Microscopy (SEM), we assessed their interaction with adipose mesenchymal stem cells. Thereafter, the in vivo behavior of scaffolds was studied implanting them in the omentum of Sprague Dawley rats. At 4 weeks, explants were processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). Considering the in vitro evidence, both histological and SEM results proved the effectiveness of the decellularization, and allowed to appreciate the preservation of intestinal villi of the wIM as well as the characteristic features of the hIM. At 7 days from cell seeding, MTT assay showed that hIM/OxPVA scaffolds could support cell adhesion/proliferation even if the wIM/OxPVA ones seem to significantly increase cell growth (p <0.01). Considering in vivo data, around the cryogels was recognizable a continuous and relatively organized tissue wall; its thickness was greater in wIM/OxPVA scaffolds than in wIM/OxPVA and OxPVA (control) ones. The presence of Ki-67+ elements, proving cell proliferation, was mainly ascribable to lymphocyte-macrophage populations and in minority to connective and myofibroblastic ones; primarily on the outer sides, CD3+ and F4/80+ cells were found. Moreover, the outer layer of the tissue wall showed a connective appearance partially immunoreactive for both anti-Desmin and -αSMA, which are related to myofibroblastic features and smooth muscle cells. In the parietal thickness, vascular structures with organized endothelium were found. Towards the polymer, cubic/cylindrical cells partially positive for anti-MNF116 were recognizable and they were ascribable to epithelial cells. Both scaffolds, albeit with some difference, are promising, nevertheless further analysis will be necessary.

Characterization of novel autologous leukocyte fibrin platelet membranes for tissue engineering applications

Autologous hemocomponents have recently emerged as potential biologic tools for regenerative purpose, consisting mainly of platelet concentrates which locally release growth factors (GFs) to enhance the tissue healing process. Despite two decades of clinical studies, the therapeutic efficacy of platelet concentrates is still controversial. This work represents a first characterization of a novel autologous leukocyte fibrin platelet membrane (LFPm), which is prepared by the Department of Immunohematology of Belluno Hospital according to a well standardized protocol. The quantification of their specific content showed that LFPms are enriched not only with platelets, but also with monocytes/macrophages, fibrinogen and CD34+ cells. Mechanical properties of LFPms were investigated by tensile tests, revealing that the specific elasticity of membranes was maintained over time. Furthermore, the release kinetics of Platelet Derived Growth Factor, Vascular Endothelial Growth Factor, Tumor Necrosis Factor alpha and Interleukin-10 was assessed by ELISA, demonstrating that LFPms act as GF delivery systems which sustain the local release of bioactive molecules. For in vitro biodegradation analysis, LFPm samples were incubated into PBS solution for 4, 7, 14, 21 days. SEM micrographs showed a progressive loss in cellular elements associated to a simultaneous exposure of the fibrin scaffold, also confirmed by histological and immunohistochemical investigations. In parallel, LFPm disks were implanted into a subcutaneous dorsal pouch of healthy nude rats and explanted after 4, 7, 14, 21 days for in vivo biodegradation study. SEM, histological and immunohistochemical analysis revealed that the typical LFPm fibrin structure was maintained until day 7, with a contemporary loss of cellular elements. From day 14, the morphology and texture of samples became less and less recognizable, confirming that a progressive biodegradation occurred. Overall, collected evidences could support the rationale for the clinical use of LFPms, shading some light on the regenerative effect they may exert after the autologous implant on a defect site.