Introduction 6 3 13 13 18 19 14 16 15 7 17 9 20 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 moment of the implantation. Materials and methods ® 6 2 Cell viability analysis 2 The results were evaluated by means of the spectrophotometric assay (570 nm), yielding absorbance as a function of viable cell number. Histochemical and immunohistochemical analysis The samples were fixed by immersion in 4% para-formaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C and then embedded in paraffin. Specimens were stained with safranin-O. For the immunohistochemistry, non-specific binding was blocked with 3% normal goat serum in a phosphate-buffered saline (PBS), pH 7.4, for 30 min at room temperature; slides were then incubated overnight with primary antibodies at 4°C. Sections were incubated with polyclonal antibodies anti S-100 protein (Dako, Italy), a cytoplasmatic marker of chondrocyte phenotype, diluted at 1:3,000, anti-collagen type I (Monosan, The Netherlands) and II (Calbiochem-Oncogene, CA, USA) at 1:150, and monoclonal antibodies anti chondroitin sulphate (chondroitin-S) (Sigma) at 1:200. Rabbit and mouse immunoglobulins, at the same dilutions as the primary antibodies, were used as controls. After three washes with Tris–HCl (0.05 M, pH 7.6), revelation of the reactions was accomplished by DAKO LSAB + kit, HRP. Stainings were viewed and photographed with a Leica Microscope (Leica Cambridge Ltd., UK). Ultrastructural analysis For scanning electron microscopy (SEM), the membranes were fixed in 2% glutaraldeyde in 0.1 M cacodylate buffer (pH 7.4), post-fixed in 1% osmium tetroxide, dehydrated in increasing ethanol concentrations and then CPD-dried. They were mounted on stubs and gold-sputtered. Specimens were observed with a Philips 505 microscope. Results Cell viability analysis 1 Fig. 1 Macroscopic view of human chondrocytes seeded on type I, III collagen membrane after MTT incubation. The converted blue dye shows the presence of numerous viable cells with quite a homogeneous stain distribution  2 1 Fig. 2 1  Table 1 2 Case Joint Age Sex Seeded cells 2 1  Knee 54 M 6 3 2  Knee 22 F 6 3 3 Knee 50 F 6 3 4  Knee 23 M 6 3 5 Knee 29 F 6 3 6 Knee 43 M 6 3 7 Knee 48 M 6 3 8 Ankle 42 F 6 3 9 Knee 19 M 6 3 10 Knee 35 M 6 3 11 Ankle 39 M 6 3 12 Knee 13 M 6 3 2 Histochemical and immunohistochemical analysis 3 4 5 Fig. 3 Human chondrocytes seeded on type I, III collagen membrane before the implantation. Numerous cells are located on the membrane and deeper in the matrix forming a multi-layer engineered tissue. (Safranin O stain, ×200)  Fig. 4 Human chondrocytes seeded on type I, III collagen membrane. A marked cytoplasmic immunoreaction for S-100 protein is evident (×400)  Fig. 5 Human chondrocytes seeded on type I, III collagen membrane. Cells express a positive immunoreaction for type II collagen, while the membrane remains unstained (×400)  Ultrastructural analysis 6 Fig. 6 arrows  Discussion Cell phenotype and proliferation analysis should be an essential step in the evaluation of all tissue-engineered products because the implantation of dedifferentiated or not-proliferating cells would not justify the therapeutic employment of these biotechnologies. 1 4 1 4 11 8 10 9 20 5 12 20 2 7