Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

Autologous implantation of chondrocytes on a solid collagen scaffold: clinical and histological outcomes after two years of follow-up

Abstract From our overall experience in 56 patients, we here report the treatment with matrix-induced autologous chondrocyte implantation (MACI) of 35 patients suffering from knee cartilage defects measuring about 4 cm², and followed for a minimum of 6 months. A total of 36 knees were treated (1 patient on both knees) and clinically observed for 22 months (in some cases for over 39 months), in accordance with a standardised protocol. Subjective parameters (pain, well-being, functional state, symptoms during specific activity) and objective outcomes (IKDC score and Lysholm and Tegner scores) were recorded. One or 2 years after implantation, some biopsies of the regenerated cartilage were histologically evaluated. The subjective parameters (VAS pain score, 2.80±1.49, p<0.0001; change vs. basal score, 2.72) promptly normalized after 1 month, as did the objective ones (IKDC score after 6 months, 1.53±0.59, p<0.0001; change vs. basal score, 1.78). Similar results were observed after the treatment of a femoropatellar kissing lesion. The three cartilage biopsies that were analysed from different patients showed a tissue positivity to immunohistochemical markers of hyaline cartilage. The conclusions of this preliminary analysis are that the clinical outcome and histological evaluation suggest that MACI is able to relieve pain and restore the functionality of the knee, and that the treatment appears capable of regenerating hyaline cartilage.     

Alpha-1-antichymotrypsin in renal biopsies

Alpha 1-Antichymotrypsin (alpha 1-AK) and alpha-1-antitrypsin (alpha 1-AT) represent a defense mechanism to protect the tissues from proteolytic enzyme activity. We studied the implication of alpha 1-AK and alpha 1-AT in glomeruli of patients with different nephropathies based on the analysis of 52 paraffin-embedded renal biopsies with alpha 1-AK and alpha 1-AT antisera. The results demonstrate an intense alpha 1-AK glomerular staining in renal biopsies from patients with minimal-change disease, while a minor staining of this protein was found in the other nephropathies. No significant evidence of alpha 1-AT deposits was observed in our cases. Our findings suggest that when alpha 1-AK is lacking in glomeruli the defense mechanisms against proteolytic enzymes may not be efficient enough to protect the glomerular structures and limit the damage. Since alpha 1-AK is a reactant of the acute phase of inflammation, it may be considered as a marker of activity for monocyte-macrophages in glomerular damage.     

Increased external tibial torsion in Osgood-Schlatter disease

We studied the relationship between Osgood-Schlatter disease and torsional abnormalities of the lower limb in 21 boys with this condition and 20 age- and sex-matched controls. 3 groups of knees (20 control knees, 21 symptomatic and 21 asymptomatic or less symptomatic knees) were subjected to clinical, radiographic and CT evaluation. We found no statistically significant differences between patients and controls, as regards femoral anteversion, patellar congruence angle, patellar tilt angle and anterior tibial tuberosity-trochlear groove distance, but the condylomalleolar angle and tibial torsion angle were greater in patients. We found no differences between symptomatic and asymptomatic or less symptomatic knees in any of the parameters. All the symptomatic knees were on the side preferentially involved in jumping and sprinting. This increase in external tibial torsion may play a role as a predisposing mechanical factor in the onset of Osgood-Schlatter disease in male athletes.     

Mitotic polyploidization in trophoblast giant cells of the alpaca

Genome multiplication is a typical feature of trophoblast giant cell (TGC) development in many species. Elevated nuclear DNA contents can be achieved by modified cell cycles with a complete lack of mitosis (endoreduplication) or with incomplete mitoses. The aim of this study is to characterize genome multiplication in the alpaca TGC. Placental tissues of gestation days 150, 264 and 347 (near term) and term placentae were processed for light microscopy and for transmission electron microscopy. Each TGC showed many nuclear profiles. Observation of serial sections revealed that TGCs are truly multinucleate with several highly lobulated nuclei. Feulgen staining showed that TGC nuclei have a higher DNA content than nuclei of other trophoblast cells. The number of argyrophilic nucleolar organizer regions (AgNORs) in nuclear profiles of TGC was between 15 and 100, while other trophoblast cells showed 1 or 2 AgNORs. Large multipolar mitotic figures with maximal diameters of 80 mum were observed in the alpaca placentas on gestation days 264 and 347. No cytokinesis was seen in TGC. The results show that the mode of genome multiplication in the alpaca TGC is mitotic polyploidization. Subsequent acytokinetic mitoses may lead to an accumulation of chromosomes and centrioles in TGC. With increasing ploidy levels, the shape of these polyploidizing mitoses becomes more irregular. The restitution of nuclei after these complex multipolar mitoses is likely to result in the irregular nuclear shape in TGC.     

Haplotype analysis of BRCA1 gene reveals a new gene rearrangement: characterization of a 19.9 KBP deletion

Germ-line mutations in the BRCA1 gene cause hereditary predisposition to breast and ovarian cancer. BRCA1 and BRCA2 mutations account for about 40% of high-risk families. Mutation-screening methods generally focus on genomic DNA and are usually PCR based; they enable the detection of sequence alterations such as point mutations and small deletions and insertions. However, they do not allow the detection of partial or entire exon(s) loss, because the presence of the homologous allele results in a positive PCR signal, giving rise to a false-negative result. Identification of unusual haplotypes in patient samples by an expectation maximization algorithm has recently been suggested as a method for identifying hemizygous regions caused by large intragenic deletions. Using a similar approach, we identified a novel BRCA1 genomic rearrangement in a breast/ovarian cancer family negative at the first mutation screening; we detected a deletion encompassing exons 14-19, probably due to replication slippage between Alu sequences.     

Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.     

Dibasic staining of large epoxy tissue sections and applications to surgical pathology

A variety of normal and pathologic aldehyde-fixed osmium postfixed human tissues were prepared as large sections embedded in Spurr epoxy. They were stained with a sequential basic fuchsin--methylene blue stain which gives "hematoxylin- and eosin-like" staining and additionally functions as several special stains. This technic also allows for electron microscopy directly on the embedded tissue. The histologic and cytologic preservation and overall staining was superior to tissue embedded in glycol methacrylate. The methods and technics presented in this article have important applications in diagnostic surgical pathology and histology in general.