Two qualitatively different stages in fibronectin production by confluent cultures of human fibroblasts are demonstrated by immunomorphological and immunobiochemical methods. In the f'trst stage fibronectin is accumulated exclusively in the cytoplasm, while excretory synthesis predominates in the second stage. The maximum production of fibronectin is observed on day 3 in culture. It is concluded that 3-day-old cultures of human fibroblasts are preferable for wound treatment. Key Words: fibronectin; fibroblastsThe treatment of extensive thermal burns with the use of cultured human fibroblasts (FB) has found wide application in clinical practice [1,[4][5][6]. The method is based on the stimulatory effect of FB on the adhesion and proliferation of epidermocy-tes. Fibronectin (FN), a high-molecular-weight glycoprotein produced by grafted FB, is a key factor in these processes. It stimulates both adhesion and proliferation of epidermocytes [2], and therefore, the level of FN synthesis should be regarded as an important determinant of the optimal times for FB transfer onto a wound. Our objective was to study the dynamics of FN synthesis in a confluent culture of human FB and to determine the times of maximum FN production. MATERIALS AND METHODSThe dynamics of FN synthesis by human FB was studied in 4th passage cultures. A primary culture was initiated from fragments of the derma [3]. Cells were cultured in Eagle's medium with 10% fetal calf serum and 2% glutamine at 37~ in a CO 2 incuba-A. V. Vishnevskii Institute of Surgery', Russian Academy of Medical Sciences, Moscow tor (Flow). Quantitative and qualitative analyses of the FN content were performed on days 1, 3, and 6 of culturing using immunomorphological and immunobiochemical methods.Fibronectin was identified by the immunoperoxidase method. Murine mononclonal antibodies against human FN (Antifibronectin, cell attachment fragment, Boehringer Mannheim, No. 1087720) were used. Cell cultures were fLxed with acetone for 10 rnin at -12~ rinsed with neutral phosphate buffer, treated with the antibody (10 mg dry matter/ml phosphate buffer), washed three times in phosphate buffer, and incubated with anti-mouse IgG conjugated with peroxidase (1:10, N. F. Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences). Incubation was carded out for 60 min at 37~ in a humidified atmosphere. The cultures were then washed three times with neutral phosphate buffer and treated with freshly prepared substrate solution: 3,3'-diaminobenzidine (Sigma) in Tris-HC1 buffer (0.5 mg/ml, pH 7.3) with 3% hydrogen peroxide. Preparations treated with nonimmune serum or with one of the monoclonal antisera served as the controls.The FN content in the culture medium and in FB was determined by the immunoturbidimetric