A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering.

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na(+)-K(+) ATPase, and FLK-1. Na(+)-K(+) ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

F-actin and α-actinin reorganization mediates initial fibroblast interaction with CoCr alloy particles in vitro.

Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F-actin and α-actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL-6 and IL-1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F-actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α-actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy-dependent process involving actin filaments. Changes in the levels of cytokine IL-6 and IL-1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts.

Hypoxia-like effect of cobalt chromium alloy micro particles on fibroblasts in vitro.

Periprosthetic osteolysis leading to asceptic loosening remains the primary cause of failure of joint replacement. Although many inflammatory cell types have been implicated, the exact pathomechanisms of asceptic loosening have not been delineated. In the present study we have adopted a proteomic approach to elucidate the initial signals that are expressed to particulate material, using an in vitro cell culture system. Human lung fibroblasts MRC-5 were cultured with Cobalt Chromium (CoCr ASTM F-75, 1-7 µm) particles. Cells were harvested after 72 h incubation and total cellular proteins extracted for downstream analysis via 2D Gel Electrophoresis and tandem mass spectrometry using MALDI-TOF-TOF-MS. Thirteen protein spots showed greater than twofold increase, following 72 h incubation of fibroblast with CoCr particles. Four of these proteins were identified by tandem mass spectrometry. These were Annexin II, Pyruvate kinase, Triose phosphate isomerase, and N-myc downstream regulated gene 1 protein. Cobalt is a hypoxia mimicking agent and N-myc downstream regulated gene 1 protein, Triose phosphate isomerase, Pyruvate kinase, and Annexin II are important hypoxia regulated gene products that are found to be over expressed in cellular oxidative stress response. Our data indicates that exposure of fibroblast to CoCr alloy induces the transition of these cells into a hypoxia like state and oxidative stress even in normoxic culture conditions. The study reflects the possibility of the presence of a hypoxic environment in the periprosthetic tissue surrounding metallic implants.