Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions

AbstractObjective To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases.Design Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification.Results Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes.Conclusions The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans

A molecular and histological characterization of cartilage from patients with Morquio syndrome

SummaryObjectiveTo investigate the gene expression profile and the histological aspects of articular cartilage of patients affected by Morquio syndrome, a lysosomal storage disease characterized by the accumulation of glycosaminoglycans within the cells which result in abnormal formation and growth of the skeletal system.MethodArticular cartilage samples were obtained from the femoral condyle of two siblings with Morquio syndrome during surgery performed to treat valgus knee. As controls, four biopsy samples of healthy cartilage were obtained from four different male multiorgan donors. A Real-Time Polymerase Chain reaction (RT-PCR) analysis was performed to evaluate the expression of type I and II collagens and aggrecan mRNAs. Histological and immunohistochemical analyses for some matrix proteins were carried out on paraffin embedded sections.ResultsType I collagen mRNA mean level was higher in the samples of patients with Morquio syndrome compared to controls. Type II collagen and aggrecan mRNAs' mean expression was instead lower. The morphological appearance of the cartilage showed a poorly organized tissue structure with not homogeneously distributed cells that were larger compared to normal chondrocytes due to the presence inside the vacuoles of proteoglycans which were not metabolized. Chondrocytes were negative for collagen II immunostaining while the extracellular matrix was weakly positive. Collagen type I immunostaining was positive at cellular level. Keratan sulfate showed diffuse positivity and chondroitin-6-sulfate was present throughout the cartilaginous thickness.ConclusionIn cartilage of patients with Morquio syndrome, a low expression of collagen type II and a high expression of collagen type I both at protein and molecular levels are evidentiated. This finding could give evidence of the reduction in ankle and knee joint movement observable in these patients

Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYAFF11): Molecular, Immunohistochemical and ultrastructural analysis.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. Such scaffolds should provide a preformed three-dimensional shape and prevent cells from escaping into the articular cavity. Furthermore, these constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and their phenotype redifferentiation. The aim of this study was to verify the ability of HYAFF®11, a recently developed hyaluronic-acid-based biodegradable polymer, to support the growth of human chondrocytes and to maintain their original phenotype. This capability was assessed by the evaluation of collagen types I, II and aggrecan mRNA expression. Immunohistochemical analyses were also performed to evaluate collagen types I, II and proteoglycans synthesis. A field emission in lens scanning microscopy was utilized to verify the interactions between the cells and the biomaterial. Our data indicate that human chondrocytes seeded on HYAFF®11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration for the therapeutic potential of HYAFF®11 as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects

Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment

(1) Background: Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection of 0.5 mu M. Finally, it can be successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H2S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Inflammatory molecules produced by meniscus and synovium in early and end-stage osteoarthritis: a coculture study

The aim of this study was to identify the molecules and pathways involved in the cross-talk between meniscus and synovium that may play a critical role in osteoarthritis (OA) pathophysiology. Samples of synovium and meniscus were collected from patients with early and end-stage OA and cultured alone or cocultured. Cytokines, chemokines, metalloproteases, and their inhibitors were evaluated at the gene and protein levels. The extracellular matrix (ECM) changes were also investigated. In early OA cultures, higher levels of interleukin-6 (IL-6) and IL-8 messenger RNA were expressed by synovium and meniscus in coculture compared with meniscus cultured alone. RANTES release was significantly increased when the two tissues were cocultured compared with meniscus cultured alone. Increased levels of matrix metalloproteinase-3\ua0(MMP-3) and MMP-10 proteins, as well as increased release of glycosaminoglycans and aggrecan CS846 epitope, were observed when synovium was cocultured with meniscus. In end-stage OA cultures, increased levels of IL-8 and monocyte chemoattractant\ua0protein-1\ua0(MCP-1) proteins were released in cocultures compared with cultures of meniscus alone. Chemokine (C-C motif) ligand 21 (CCL21) protein release was higher in meniscus cultured alone and in coculture compared with synovium cultured alone. Increased levels of MMP-3 and 10 proteins were observed when tissues were cocultured compared with meniscus cultured alone. Aggrecan CS846 epitope release was increased in cocultures compared with cultures of either tissue cultured alone. Our study showed the production of inflammatory molecules by synovium and meniscus which could trigger inflammatory signals in early OA patients, and induce ECM loss in the progressive and final stages of OA pathology