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.

Detection of viral DNA in kidney graft preservation and washing solutions is predictive of posttransplant infections in pediatric recipients.

BACKGROUND: In pediatric kidney transplant recipients, viral infections occur soon after transplant and may be transmitted from the graft. METHODS: This study of 75 pediatric kidney transplants investigated whether genome sequences of parvovirus B19, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and BK polyomavirus (BKV) could be detected in kidney graft samples (graft biopsy samples and preservation and washing solutions) collected before implantation and whether their presence was a risk factor for infections in the recipient. RESULTS: B19 DNA was detected in approximately 30% of graft biopsy samples, preservation solutions, and washing solutions; EBV DNA was detected in approximately 20% of preservation and washing solutions but rarely in biopsy samples; and HCMV DNA and BKV DNA were rarely detected in graft biopsy samples. Seronegative recipients of B19 DNA-positive and EBV DNA-positive grafts had a significantly higher risk of infection during the early posttransplant period than did recipients of negative grafts. In particular, none of the B19-seronegative recipients of B19 DNA-negative grafts experienced infection soon after transplant, whereas most recipients of B19 DNA-positive grafts experienced infection within the first month after transplant. CONCLUSIONS: Molecular testing of donor grafts for viruses that infect circulating and resident cells in the graft-such as B19 in the kidney-could be useful (in association with donor/recipient serostatus) for identifying recipients at high risk for posttransplant infections.

The effect of donor/recipient body surface area ratio on outcomes in pediatric kidney transplantation.

In pediatric kidney transplantation, the effect of inadequate nephron dosing on graft survival remains undetermined. The aim of this study was to assess the use of D/R BSA, as a reliable indicator of adequate nephron dosing, and eventually a tool to optimize pediatric graft allocation. Following Institutional Review Board approval, we reviewed deceased donor pediatric kidney transplantation (N = 156). We divided patients into three groups, based on D/R BSA: A < or =0.8; B 0.81-1.19; C > or =1.2. Five-yr graft survival rates in the groups were: A 82.0%; B 94.9%; C 97.1% (p = 0.01). Group C had the lowest rate of acute rejection, suggesting a protective effect of increased D/R BSA (group A = 35.7%, group B = 38.9%, group C = 18.8%; p = 0.029). The logistic regression analysis showed that decreased D/R BSA ratio is a risk factor for loss of graft function, at one and five yr [i.e., group A OR 6 (95% CI 1.14-39.30, p = 0.015) and OR 4.49 (95% CI 1.46-13.79, p = 0.009), respectively]. We conclude that for pediatric recipients, D/R BSA is a valuable adjunct when determining long-term graft survival. Its utility may avoid an alloimmune-independent risk factor, increasing the long-term protective value of a good matching policy.

Efficient delivery of human single fiber-derived muscle precursor cells via biocompatible scaffold.

The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

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 © 2015 John Wiley & Sons, Ltd.

Myoblast-acellular skeletal muscle matrix constructs guarantee a long-term repair of experimental full-thickness abdominal wall defects.

To obtain a valuable treatment of congenital muscle defect, cell-matrix constructs composed of satellite cell-derived myoblasts (XY karyotype) seeded on muscle acellular matrices were used to repair a previously created full-thickness defect of abdominal wall of 18 1-month-old female Lewis rats. Acellular abdominal matrices, obtained by a detergent-enzymatic method, were positive for both basic fibroblast growth factor and transforming growth factor-beta, and were able to support in vitro cell adhesion. All animals survived the surgery, without signs of infection or implant rejection, and were humanely killed at 1, 3, or 9 months after surgery. The implants appeared well preserved, were integrated in the host tissue, and maintained their original dimension and thickness until 9 months. Vesicular acetylcholine transporter was expressed on the surface of muscle fibers from 1 month postsurgery. Finally, implanted male myoblasts were present inside the patches until 9 months, as demonstrated by the expression of SrY mRNA and by the presence of Y chromosome probe signal. These results allow us to conclude that cell-matrix constructs could represent a promising approach to the repair of muscle defects, because they are repopulated in vivo by skeletal muscle cells and nervous elements and maintain their structural integrity over the long term.

Homologous muscle acellular matrix seeded with autologous myoblasts as a tissue-engineering approach to abdominal wall-defect repair.

Myoblasts were obtained by culturing in vitro, single muscle fibers, isolated by enzymatic digestion from rat flexor digitorum brevis, and their phenotype was confirmed by myogenic differentiation factor, myogenic factor-5, myogenin and desmin. Cultured myoblasts were harvested and seeded on patches of homologous acellular matrix, obtained by detergent-enzymatic treatment of abdominal muscle fragments. Myoblast-seeded patches were inserted between obliqui abdominis muscles on the right side of 1-month-old rats, while non-seeded patches were implanted on the left side. Thirty days after surgery, non-seeded patches were completely replaced by fibrous tissue, while the structure of myoblast-seeded patches was well preserved until the 2nd month. Seeded patches displayed abundant blood vessels and myoblasts, and electromyography evidenced in them single motor-unit potentials, sometimes grouped into arithmic discharges. Ninty days after implantation, the thickness of myoblast-seeded patches and their electric activity decreased, suggesting a loss of contractile muscle fibers. In conclusion, the present results indicate that autologous myoblast-homologous acellular muscle matrix constructs are a promising tool for body-wall defect repair, and studies are under way to identify strategies able to improve and maintain the structural and functional integrity of implants for longer periods.

[Antireflux surgery in neurologically impaired children]. / La plastica antireflusso nel paziente pediatrico neuroleso.

OBJECTIVE: To evaluate results of Nissen fundoplication in a neurologically impaired children population versus normal, considering symptoms improvement, general conditions, parents satisfaction, facility to assist the patient. METHODS: 57 patients were analysed, 38 neurologically impaired children (NL),19 neurologically normal children (NNL), that underwent fundoplication during six-year period (Feb '95-Dec '00). Mean age at surgery was 6,8 years (range 3 month- 18 years) for NL; 4,7 years for NNL (1 month- 15 years). We contact 31 family (19 NL, 12 NNL) before and after surgery and we subject them a questionnaire. For data analysis chi-squared test and Mann-Witney Rank Sum test has been used. RESULTS: A significantly greater reduction of the vomit and regurgitation have been observed in both groups (p<0.5). Drooling was significantly reduced only in the NNL group (p<0.5) than in the NL group. Cough doesn't improved significantly in both groups (p>0.5). Major respiratory symptoms and respiratory infections improved significantly only in NL group (p<0.5). The parents satisfaction is high and patients management results easier. CONCLUSIONS: The Nissen fundoplication in NL children is an effective procedure in order to obtain improvement on vomit; major respiratory symptoms and quality of caregivers management.

Effect of cryopreservation on in vitro clonal growth of cordonal blood cells.

Cordonal blood (CB) is today recognized as a potentially important source of hematopoietic stem cells (SCs) for allogeneic transplantation, and to this task it would be of extreme importance to have the possibility of using cryopreserved CB units. Hence we investigated whether freezing and thawing alter the viability of CB hematopoietic cells. Mononuclear cells, recovered from fresh CB units by density-gradient centrifugation, were partly frozen and then thawed and partly immediately utilized for clonogenic tests. The cells were cultured in H4330 (C group) or H4434 (H4330 added with SC clonogenic growth factors) (C+ group) semisolid media added or not with serum-free Dulbecco modified Eagle medium (DMEM/sf). Cells seeded on H4330 were exposed to the conditioned supernatants from two human-embryo liver cell lines, which have been previously found to stimulate clonal growth of fresh CB hematopoietic cells. As expected, the number of colony-forming units (CFU) was higher in C+ than C group, and was not influenced by the addition of DMEM/sf. CFU number was higher in cryopreserved than fresh cells in both C and C+ culture groups. Conditioned supernatants from both cell lines stimulated clonal growth in both fresh and cryopreserved cell cultures. These findings indicate that cryopreservation and thawing do not alter the viability of CB SCs, but, on the contrary, improve their basal and cytokine-stimulated clonal growth, probably by negatively selecting SCs among the mononuclear CB cell population.

New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells.

We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM-CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder-layers for hematopoietic CB SC expansion in vitro.

Anorectal malformation and associated end-stage renal disease: management from newborn to adult life.

BACKGROUND/OBJECTIVE: Renal failure remains one of the most significant causes of morbidity in patients with anorectal malformations (ARM). In the modern era, an increasing number of children born with ARM and genito-urinary (GU) anomalies reach adulthood and require continued multidisciplinary care for the rest of their life. The aim of this study is to present our institutional experience in the management of pediatric chronic renal failure related to severe GU anomalies and anorectal malformations. METHODS AND RESULTS: Three hundred twenty-one patients with ARM have been followed at our institution since 1987. Six patients developed end-stage renal disease (ESRD) and received a kidney transplant at different ages. One patient is currently followed for mild, progressive chronic renal failure. These seven cases are reported along with a broad discussion concerning etiology of renal failure, neonatal surgical management, pediatric dialysis, urologic issues, and kidney transplantation. CONCLUSION: Complex GU anomalies associated with ARM require a long-term approach by specialized pediatric and adult clinicians to optimize the care of this selected population of patients.

An unusual case of acute pancreatitis and gastric outlet obstruction associated with wandering spleen treated by laparoscopic splenopexy.

Wandering spleen (WS) is an uncommon condition, usually asymptomatic, often recognized as an incidental finding. When symptoms occur, they can vary, although acute abdominal pain is the most common presentation in the pediatric population. In some cases, WS can become a dangerous condition because of the risk of splenic ischemia from persistent pedicle torsion. We describe a case of WS in a 3-year-old boy presenting with vomiting, abdominal swelling, and acute pancreatitis; the diagnosis was obtained by ultrasound and computed tomography. Laparoscopic splenopexy was successfully performed through an extraperitoneal pocket and a Vicryl mesh. To the best of our knowledge, the combination of gastric outlet obstruction and acute pancreatitis has never been reported as presenting symptoms of WS.

Murine muscle precursor cells survived and integrated in a cryoinjured gastroesophageal junction.

BACKGROUND: Mini-invasive techniques for gastroesophageal reflux disease (GERD), such as endoscopic injections of inert materials, have been introduced in recent years. However, results are still preliminary. Cell injection has emerged as an alternative strategy in both vesicoureteral reflux and incontinence. Here we report, for the first time, the injection of muscle precursor cells (MPCs) in the gastroesophageal junction (GEJ). MATERIALS AND METHODS: MPCs were derived from expanded satellite cells isolated from skeletal muscle fibers of green fluorescent protein (GFP) positive mice. Via laparotomy, GFP-negative mice were subjected to cryoinjury of GEJ followed by injection of MPCs (experimental animals), bone marrow derived cells, or saline (controls). RESULTS: Immunofluorescence analyses of experimental GEJs demonstrated coexpression of GFP and desmin in grafted cells. GFP+ muscle neofibers were evident at 4 wk after injection. Coexpression of GFP and smooth muscle actin was also observed at 2 wk. CONCLUSIONS: Satellite cells could be easily harvested, expanded in culture, and used as injectable substance in the GEJ. These results could be the background for the development of a new injection technique for GERD treatment, which might combine bulging and functional actions.