Hamstring grafts are tenogenic constructs for ACL reconstruction and Pulsed Electromagnetic Fields improve tendon specific markers expression. An in-vitro study.

Hamstring tendons represent one of the commonest autologous graft used during ACL reconstruction. The harvest of the tendon and the time of tendon processing on the operating table, together with the pretensioning maneuvers and the permanence out of the joint during the time of surgery, might impair tendon derived cells (TCs) viability. The aim of the study was: i) to assess the effective viability of the TCs at the end of the surgical procedure; ii) to investigate if TCs viability and the expression of tendon specific markers may be improved through exposure to prolonged pulsed electromagnetic fields (PEMF) similar to that of clinical practice. Remnants of semitendinosus and gracilis tendons (discarded at the end of the ACL reconstruction) were collected from 13 healthy donors. To isolate TCs, the tendon tissue was minced and digested enzymatically with 0.3% type I collagenase in DMEM with continuous agitation for 15 h at 37°C. The isolated nucleated cells were then plated at 5x103 cells/cm2 in a complete medium composed of DMEM, 10% fetal bovine serum, 50 U/ml Penicillin, 50 mg/ml Streptomycin, 2 mM L-glutamine, and supplemented with 5 ng/ml basic fibroblast growth factor (b-FGF). They were maintained at 37 °C in a humidified atmosphere with 5% CO2, changing culture medium every 3 days. When they reached 80-90% of confluence, the cells were detached by incubation with trypsin/EDTA and then cultured at a density of 5x103 cells/cm2. TCs were cultured in complete medium for 7, 14, 21 days (in chamber slides, to optimize the final immunofluorescence analysis). The following cell cultures were set up: i) TCs cultured with differentiation medium + exposure to PEMF 8 h/day; ii) TCs cultured with differentiation medium without exposure to PEMF. The stimulation with PEMF was generated by a pair of electrical coils, connected with the generator of pulsed electromagnetic fields (PEMF generator system IGEA, Carpi, Italy, intensity of magnetic field = 1.5 mT, frequency = 75 Hz). At day 0, day 7, day 14 and day 21 immunofluorescence analysis was performed to evaluate the expression of tendon specific markers (collagen type I, collagen type VI, scleraxis) and proliferative markers (PCNA, beta-catenin). The TCs from the hamstring tendon fragments at the end of the ACL reconstruction were alive and they expressed markers of proliferation and tendon phenotype at the end of the culture period. The TCs in the presence of PEMF 8h/day showed a greater production of collagen type I, collagen type VI and scleraxis than TCs cultured without PEMF (p<0.05). The expression of these markers increased from 7 to 21 days of culture. The expression of proliferative markers in the presence of PEMF stimulus was significantly lower (p<0.05) than that of TCs cultured without PEMF. Hamstring tendons are not simple "tenoconductive" scaffolds but biologic alive tenogenic constructs rich in cells that can sustain tenogenic behavior and tendon matrix synthesis. Prolonged exposure to PEMF improves their phenotype. Thus, from a clinical perspective, the use of PEMF may represent a possible future strategy to positively influence the early phase of graft remodeling and, ultimately, improve the ligamentization process. Following these concepts, further studies might also exploit the anabolic role of PEMF as an adjunctive postoperative strategy in different tendon pathologies.

Tips and tricks for writing a scientific manuscript.

An original scientific manuscript is the target for any researchers whose aim is to show the innovative results arising from the original intuitions that drove all their experiments. Time and patience are essential to decide how to present the data, how to conceive the tables and figures representing the main outcomes of the research, and how to read and mention the necessary references. Few basic rules may help in this difficult task. The first basic rule is: "do not follow the sequence of the paper". On the opposite, i) start writing the "Materials and Methods (or Patients and Methods when dealing with a clinical study)", ii) then write the "Results" section, iii) then, write the "Discussion" paragraph, in which the principal investigator explains the results and the innovations proposed, iv) then, write the "Introduction", which should be clear and concise. The last element to be written should be the "Abstract", which is the "interface" between the authors and the readers. The second basic rule is that any of the central chapters of the manuscript, i.e. "Materials and Methods" (MM), "Results" (R) and "Discussion" (D), should follow a methodical and sequential description of the topics in a "corresponding sequence of paragraphs". In other words, in the R and the D chapter sequence of the paragraphs should be linked to the sequence of the concepts described and discussed in the paragraphs of the MM chapter. Thus, a sequential description of concepts will be easily followed by the writers, facilitating both the authors in the organization of the data and the reader in finding a reasonable "answer" to all the aspects of the study mentioned in the MM chapter. In this article, these two rules are extensively described and several tips and tricks for each chapter are suggested to ease the composition of a scientific paper. Indeed, it may be possible to solve the complex problem of "writing a scientific paper" by means of separating it in main sections (chapters) and subsections (paragraphs) and dealing with them one by one. Naturally, this takes time and passion, but, as affirmed by Steve Jobs, "the only way to do great work is to love what you do".

One-step cartilage repair with minced chondral fragment on a composite scaffold: an in vitro human study at low oxygen tension.

Minced cartilage fragments are a viable cell source for one stage cartilage repair. However, the joint surface is a low oxygen tension microenvironment and little evidence is present in literature regarding the behaviour of cartilage fragments in this peculiar condition. The aim of the study is i) to verify if low oxygen tension could negatively influence chondrocyte outgrowth from cartilage fragments into a Hyaluronic-Acid(HA)/fibrin scaffold and ii) to evaluate its effects on the behaviour of migrating chondrocyte, compared to normoxic condition. A slight decrease in chondrocyte migration and proliferation was observed in low oxygen tension cultures. Conversely, an increase in the expression of SOX9, ß-catenin, HIFs, collagen-I and II (p<0.05) in migrating chondrocytes from low oxygen tension cultures was present. Thus, a long term- exposure at low oxygen tension seems to improve the chondrocytic phenotype expression of cell outgrowing from cartilage fragments onto a HA/fibrin scaffold.

Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells as a Potential Source for Cartilage and Bone Regeneration: An In Vitro Study.

Umbilical cord (UC) may represent an attractive cell source for allogeneic mesenchymal stem cell (MSC) therapy. The aim of this in vitro study is to investigate the chondrogenic and osteogenic potential of UC-MSCs grown onto tridimensional scaffolds, to identify a possible clinical relevance for an allogeneic use in cartilage and bone reconstructive surgery. Chondrogenic differentiation on scaffolds was confirmed at 4 weeks by the expression of sox-9 and type II collagen; low oxygen tension improved the expression of these chondrogenic markers. A similar trend was observed in pellet culture in terms of matrix (proteoglycan) production. Osteogenic differentiation on bone-graft-substitute was also confirmed after 30 days of culture by the expression of osteocalcin and RunX-2. Cells grown in the hypertrophic medium showed at 5 weeks safranin o-positive stain and an increased CbFa1 expression, confirming the ability of these cells to undergo hypertrophy. These results suggest that the UC-MSCs isolated from minced umbilical cords may represent a valuable allogeneic cell population, which might have a potential for orthopaedic tissue engineering such as the on-demand cell delivery using chondrogenic, osteogenic, and endochondral scaffold. This study may have a clinical relevance as a future hypothetical option for allogeneic single-stage cartilage repair and bone regeneration.

Adhesion prevention in ventral hernia repair: an experimental study comparing three lightweight porous meshes recommended for intraperitoneal use.

BACKGROUND: In ventral hernia repair, when prosthetic material is placed intraperitoneally, it may lead to an inflammatory reaction resulting in adhesions between the mesh and abdominal viscera. Several meshes have been developed to minimize this process. In this experimental study, the ability of different combined meshes to attenuate the adhesion formation was examined. METHODS: Three commercially available lightweight porous combined meshes were placed intraperitoneally to repair an abdominal wall defect in rats: DynaMesh-IPOM (PVDF + PP), TiMesh (titanium-coated filament PP) and C-QUR/FX (omega-3 fatty acid-coated filament PP). The DynaMesh-CICAT (PVDF) was implanted in the control group. Adhesion formation was macroscopically evaluated and scored after 7 and 21 days. RESULTS: All animals except two presented intra-abdominal adhesions. None of the meshes examined in the study demonstrated to prevent adhesions. C-QUR/FX reduced adhesion formation at 7 days' follow-up compared with all other meshes but by 21 days this effect was diminished. Between 7 and 21 days adhesion extension significantly decreased for TiMesh. TAS did not show significant modifications between 7 and 21 days' follow-up for each mesh. CONCLUSIONS: The combined porous meshes tested in the present study demonstrated to reduce but not to prevent the adhesion formation, even if with some differences. Combined porous meshes could be chosen instead of simple meshes for retro-rectus preperitoneal prosthetic ventral hernia repair.

Tranexamic acid effects on cartilage and synovial tissue: an in vitro study for a possible safe intra-articular use.

The possible toxic effects of intra-articular tranexamic acid (TA) are still debated. The aim of this study was to evaluate TA effects on human cartilage fragments and synovial biopsies. Explant culture of minced articular cartilage underwent prolonged TA exposure. Histological analysis, immunofluorescence and colorimetric assay for quantification of s-GAG and DNA were performed at the end term. Synoviocytes were cultured for 48h in presence of TA. Light microscopy and flow cytometry analysis were performed at the end of the exposure to TA and one week after the treatment. TA exposure did not influence i) the chondrocyte outgrowth and migration, ii) the expression of chondrogenic and proliferative markers and iii) the s-GAG/DNA ratio. TA treatment did not affect synoviocytes' morphology and treated cells were phenotypically similar to control cells. This study demonstrated that TA does not negatively affect chondrocytes and synoviocytes cultured in vitro. Thus, our findings may be clinically relevant in order to validate the intra-articular TA administration during orthopedic procedures.

Minced umbilical cord fragments as a source of cells for orthopaedic tissue engineering: an in vitro study.

A promising approach for musculoskeletal repair and regeneration is mesenchymal-stem-cell- (MSC-)based tissue engineering. The aim of the study was to apply a simple protocol based on mincing the umbilical cord (UC), without removing any blood vessels or using any enzymatic digestion, to rapidly obtain an adequate number of multipotent UC-MSCs. We obtained, at passage 1 (P1), a mean value of 4, 2 × 10(6) cells (SD 0,4) from each UC. At immunophenotypic characterization, cells were positive for CD73, CD90, CD105, CD44, CD29, and HLA-I and negative for CD34 and HLA-class II, with a subpopulation negative for both HLA-I and HLA-II. Newborn origin and multilineage potential toward bone, fat, cartilage, and muscle was demonstrated. Telomere length was similar to that of bone-marrow (BM) MSCs from young donors. The results suggest that simply collecting UC-MSCs at P1 from minced umbilical cord fragments allows to achieve a valuable population of cells suitable for orthopaedic tissue engineering.

Seroprevalence of Toxocara infection in children and environmental contamination of urban areas in Paraná State, Brazil.

The objectives of this study were to evaluate the contamination by eggs of Toxocara in sandy areas or grass lawns of outdoor recreation areas that are used by children, and the frequency of seroprevalence in children, from three cities of fewer than 45,000 inhabitants in Paraná, Brazil. From May 2005 to December 2007, five samples were taken from each of 13 sandy sites and 18 grass lawns, all from plazas and public schools. Blood samples from children aged 0-12 years were analysed by immunoassay for anti-Toxocara IgG. The soil samples were processed by floatation and sedimentation. Eggs of Toxocara spp. were present in 44.7% (38/85) of the samples from grassed areas and in 21.4% (15/70) of the sand samples. The lawns were 2.16 times more contaminated than the sand (P = 0.0009). However, the epidemiological variables showed no statistically significant difference between seropositive (36.8%; 130/353), and seronegative children. The rate of seropositivity was higher in children aged 0-5 years (P = 0.03), who were 1.94 times more likely to develop persistent wheezing (P = 0.02).

An atypical presentation of Crohn disease in the elderly. A case report and Literature review.

The Authors present a case of an insidious onset of Crohn Disease (CD) in an elderly patient. Diagnosis complicated by extraintestinal manifestation properly of old age could be delayed and often made after surgery on the histological specimen as in our case. CD is uncommon as primary manifestation in old age, often unsuspected, incorrectly diagnosed and in many case the clinical features may lead to late diagnosis. Differential diagnosis of CD in elder people with fever, diarrhoea and abdominal pain is difficult and other symptoms affecting intestinal tract can closely mimic CD symptoms, although the pattern of clinical presentation in older patient resemble those in younger.

Multiple viral infections in HIV-infected children with chronically-evolving hepatitis.

Hepatic involvement was investigated in 31 children with perinatal HIV-1 infection, who were followed for 2-82 months (mean 30.5). Liver disease, as revealed by increased aminotransferase levels, liver biopsy or necroscopy, was diagnosed in 18 children (58%), of which 7 (22.5%) had acute hepatitis and 11 (35.5%) showed chronic liver disease. Overall, 40 persistently active or recurrent viral infections, as demonstrated by positive culture and/or detection of serum DNA, specific IgM, IgA and high levels of IgG, were revealed in the children with liver disease, while 12 similar infections were detected in 13 children without liver disease (p < 0.001). In particular, the children with liver disease showed a significantly (p < 0.002) higher incidence of cytomegalovirus (CMV) infections than children without liver disease (13 versus 3). Moreover, hepatitis C and B virus infections were revealed only in children with liver disease (5 and 1 patients, respectively). Clinical outcome showed a significantly (p < 0.001) higher mean survival in the children without liver disease than those with liver disease (47.5 versus 18.2 months). In fact, nine of the children with liver disease (50%) died, as opposed to only one of the children without liver disease (7.7%; p = 0.01). Based on these findings, liver disease is indicative of a poor prognosis in children with HIV infection, being related to the presence of multiple active viral infections.

Hepatitis in pre-school children: prevalent role of cytomegalovirus.

Virological and serological investigations were performed on 8 children with clinical and/or laboratory signs of hepatitis. Cytomegalovirus (CMV) appeared as the most frequently involved etiologic agent, since it was associated with 5 severe or chronically-evolving cases. Out of the other 3 patients with non-CMV associated hepatitis, all completely recovering, two had clinically typical Epstein-Barr virus infections, while the remaining patient had an asymptomatic HBV infection.

Hepatitis in children with thalassemia major.

Since thalassemia major patients are transfusion dependent, they are at a particularly high risk of contracting post-transfusion hepatitis. In this study, 36 transfusion-dependent children were followed up for evidence of viral hepatitis. Of 23 with increased ALT levels, 17 were anti-CMV and 12 were anti-HCV positive, 9 were positive for both CMV and HCV. Of 13 children with normal transaminase levels, 5 were CMV positive and 3 were HCV positive. These results show that CMV may be a very common cause of non-A, non-B hepatitis in transfusion dependent thalassemic children.

Acute hepatitis in childhood: virological, immunological and clinical aspects.

Virological, immunological and clinical findings in 7 previously healthy children, aged 18 months to 11 years, with viral hepatitis are reported. Asymptomatic and fully recovering, although protracted, hepatitis B was diagnosed by chance in a 1 1/2 year-old boy. Anicteric and short-term hepatitis occurred in three children with Epstein-Barr virus infection, concomitantly with typical mononucleosis syndrome. On the contrary, cytomegalovirus (CMV)-associated hepatitis was severe and protracted in two children, and fatal in a 4-year-old girl, whose main autoptic finding was submassive hepatic necrosis. Therefore, our study showed that acute viral hepatitis in non-immunocompromised children is generally self-limited and that CMV hepatitis is more frequent and severe than commonly believed.