Experimental Studies On Survival Of The Intradermally Injected Mouse Autologous Skin Fibroblasts Using Two-photon Fluorescence Microscopy

Background:The plastic surgeon are looking forward to the ideal soft tissue fillers for the treatment of facial rhytids and dermal depressions..The clinical application of synthetic and xenogenic fillers is limited because of immunologic rejection or absorption. Autologous fibroblasts cultured in vitro appears to be a living, cellular, dynamic filler system capable of immediate correction and continued repair of dermal and superficial subcutaneous deficiencies. However,deep researches on survival of the intradermally injected fibroblasts have so far been limited.Objective: Through this research, we expect to establish a fairly ideal method of isolation, cultivation, identification and transplantation of autologous mouse skin fibroblasts in vitro. On the basis of green fluorescent protein labeling and two-photon fluorescence microscopy, the current study investigated the survival profile of the intradermally injected mouse autologous skin fibroblasts, acquired the changes of the collagen fibers in the extracellular matrix,and supplies reliable theoretical basis for the clinical application of autologous skin fibroblasts for soft tissue augmentation.Methods:The primary cells were isolated from mice skin explants by attached to the wall of culture flask directly,and purified by enzymatic digestion combined with repeated cell attachment. Cellular morphologies were observed by inverted phase contrast microscope, and vimentin immunocytochemical staining and chromosome analysis were adopted as identify methods. Then the cultured cells were infected by EGFP lentivirus.Harvested the successfully labeled cells and injected them into the corresponding mouse skin, while the symmetry position were injected the same dose NS for negative control. Biopsy specimens were taken from the animals after 1 and 2 months. The specimens were made serial frozen sections,observing the survival profile of the injected cells and the changes of the collagen fibers by two-photon fluorescence microscopy. The collagenic area ratio and dermal thickness were measured by image analysis software, statistical analysis was also carried out.Results:1. Morphological observation revealed that the culture cells were spindle or irregular triangle which were arranged in a whorled or radial architecture. Immunocytochemistry results showed the passaged cells were positive for vimentin. Chromosome analysis showed the normal chromosome number in cultured cells.2. The efficiency of fibroblast cells infected by lentivirus was over 80%.After infection,the cells were still with normal appearance and reproductive activity. The fluorescence intensity of the passage cell had no attenuation.3. The nucleus was large and stained dark,with two dark nucleolus.Collagen fibers arranged disorder. Two-photon fluorescence microscopy showed clear images of the injected cells and collagen fibers. No change was found about the area of collagen fibers and the dermal thickness in 1 mouth (p>0.05);howener, the two measure values both increased after 2 months (p<0.05).Conclusions:1. Explants culture was a convenient and economic method to get skin fibroblast cells. The acquired cells could be cultured in stable condition in vitro. It was easy to obtain sufficient and reliable target cells for the study of the transplantation.2. Lentivirus vector was an ideal gene transfer vector ,because it could stably transfer the dermal fibroblast cells, with a high efficiency and no cellular injury.3. Autologous cultured fibroblast could survive in a long time after transplantating into the skin, and collagen could be newly produced, so the depth of dermis increase, which provide a possibility to treat the tissue mini-deficiencies.