Applicability of human osteoarthritic chondrocytes for in vitro efficacy testing of anti-TNFα drugs.

In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-α) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-α to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-α-treated OACs were later utilized for efficacy testing of anti-TNF-α drugs infliximab and etanercept and both significantly reduced the expressions of all catabolic and inflammatory genes tested.

Review of calciphylaxis and treatment of a severe case after kidney transplantation with iloprost in combination with hyperbaric oxygen and cultured autologous fibrin-based skin substitutes.

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is a rare complication in patients with end-stage renal disease as well as in patients after renal transplantation. It should be suspected in patients with typical painful violaceous skin lesions on the extremities or on the trunk. Active multidisciplinary management approach, with intensive local wound care, is vital in these patients. Controlling parathyroid hormone, hyperbaric oxygenation, sodium thiosulphate, bisphosphonates, cinacalcet and skin grafting could be effective. In our report, we describe a case of CUA in a 43-year-old patient two years after kidney transplantation. Despite intensive standard treatment, his wounds progressed; therefore, we decided to use iloprost, in combination with hyperbaric oxygenation. The clean wounds were then covered with cultivated autologous skin cells to enhance wound epithelialization. Seven months after finishing iloprost and hyperbaric oxygen treatment and the first application of skin substitute, the wounds healed completely and remained healed during the four-yr follow-up period. We conclude that in patients with severe CUA-induced wounds, the combined treatment with iloprost, hyperbaric oxygen and autologous cultured fibrin-based skin substitutes can be effective. A combination of different treatment modalities is vital in patients with CUA.

Evaluation of a fibrin-based skin substitute prepared in a defined keratinocyte medium.

The purpose of this study was to evaluate the influence of fibrin glue and aprotinin on the growth of adult human skin keratinocytes in defined serum-free conditions. The keratinocytes were cultured on cell culture plastics and on a fibrin matrix prepared from fibrin glue. The cell growth was measured by MTT assay, while the growth of clonogenic keratinocytes was evaluated by colony assay and expressed as colony-forming efficiency (CFE). The clonogenic potential of keratinocytes released from subconfluent and confluent cultures grown on fibrin glue was also studied by the colony assay. In comparison to a plastic culture surface the fibrin glue had significantly (P<0.05) increased the clonogenic potential of keratinocytes, as well as enhanced their growth. Keratinocytes released from subconfluent cultures grown on fibrin glue attained a significantly (P<0.05) higher percentage of clonogenic cells than their confluent parallels. At 75, 150, 300 and 450 KIU/ml aprotinin did not influence the growth of keratinocytes (P>0.2). A fibrin-based skin substitute produced in the defined keratinocyte medium could be safely used to treat a number of skin defects.