Abstract. Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confnement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-rim colloidal gold conjugated to anti-fluorescein (anti-H). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (Da) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 • 10 -9 cm2/s. These values were lower than the average diffusion coefficients (Dr) (5.4 to 9.5 x 10 -9 cm2/s) obtained by FRAP. The lower D6 is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased Do to 2.8 x 10 -9 cm2/s, but did not affect Dr.Pericellular matrix viscosity was estimated from the frictional coefficients computed from DQ and Dr and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise.To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacemerit (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 #m in diameter.

A neocartilage construct readily amenable to microscopy and biomechanical studies is described. Porcine articular cartilage was digested with a mixture of dispase and collagenase for chondrons or pronase and collagenase for chondrocytes. Chondrons or chondrocytes plated in 96-well plates were fixed and immunolabeled in situ for fluorescence microscopy at days 4 and 11. Collagen types I and II, aggrecan, and MMP-13 expression was assayed by semiquantitative RT-PCR. Cell numbers were analyzed by MTT assay. Chondrons and chondrocytes produced neocartilage that could be handled with minimal tearing on day 3 and none on day 11. Some cell division occurred between days 4 and 7. In both cultures, chondrocytes were surrounded by a thin rim of type VI collagen and osteopontin. Type II collagen, keratan sulfate, and tenascin were abundant throughout. At day 3, cells were rounded but by day 11 flattened cells were visible in the substratum. Continued synthesis of aggrecan and type II collagen mRNA indicated maintenance of the chondrocyte phenotype. The neocartilage was easy to immunolabel in situ without the need for sectioning, and individual cells were readily observed by microscopy. The versatility of these constructs makes them ideal for microscopy and for biomechanical studies.

No abstract

No abstract