Analysis The Efficacy And Safety Of SPIO And LV-GFpS As TSCs Cell Tracking Marker

Background and objectives:Tendons after acute rupture usually can not be repaired to the level of normal tendontissue. The applying method of scar formation and scar adhesion would lead to problems inhistological and biomechanical aspects after tendon damage. Tendons play an importantrole in movements, but method to improve reparation of damaged tendons remains hard.Tendon stem cells (TSCs) is a kind of progenitor cells originated from tendon tissues whichhave the potential ability of proliferation. In the normal situation, it would differentiate intotendon cells and helps to self-repair and self-replicate during the tendon damage. However,it is a little bit weak and ineffective. While in this circumstances, large amount ofexogenous TSCs applying can be an important way for injured tendons treatment.This study is modeled on rats. At the very beginning, we established the culture systemof TSCs in vitro and improved the method of obtaining, so that this will provide thefoundation for further experiment. We also explored the right mark conditions for differentcell marking methods and their affects for TSCs viability and proliferative capability invitro. In this case, we are able to provide reliable and effective cell tracking techniques forthe experiment of TSCs using in animals. After modeled acute Achilles tendon rupture inrats, we selected the right markers to mark the foreign TSCs and implanted them. Weobserved foreign TSCs’s distribution and fate in the models and certified exogenous TSCseffect on improving tendon heals in order to provide experimental foundation and theorygist in the further treatment of tendon ruptures with stem cells.Methods:1. Based on reports abroad, we adopted and improved the method of enzyme digestionto isolate and culture TSCs of rats, and observe their morphology, their cloning and cycle.We use the methods of Flow Injection Analysis, immunofluorescence, multi-induceddifferentiation and so on to indentify those TSCs,. 2. After marking the TSCs with dextran-wrapped SPIO and green fluorescent protein,we observe the effective rate of marking TSCs when using different concentrations;detection efficiency of MRI after vitro labeling and its influence in TSCs’s growth curve.We explore different MOI value’s (Lv-GFP) effect on TSCs’s marking effective rate, onpassaged attenuation when labeled in vitro, and their influence in TSCs’s stemness. Amongall these records and observations, the best suited marker and concentration would beselected to label TSCs in the following experiments.3. Model an acute Achilles tendon rupture on rat, and then inject the TSCs into the injuredlocation. The condition should be observed in the early period of injury (one week) and afterscar reconstruction (4weeks). Observe the positive effects brought by TSCs in the migration,colonization, differentiation aspects, and also the benefits to tendon healing histology.Results:1. We successfully established the way of isolating and culturing the rat tendon stemcells. Its morphology, clone ability and proliferative capability all have common charactersof stem cells. Type I collagen, stage-specific embryonic antigen (ssea)-4, Oct4expresspositive. Type III collagen is negative. Stem cell surface markers CD90, CD44are positive.CD34, CD106are negative. TSCs inducted in vitro have the obvious abilities ofosteogenesis, adipogenesis and chondrogenesis2. Detran-wrapped SPIO can mark TSCs. The effective rate rises when concentrationof SPIO goes up. When the concentration goes up to8ug/ml, the labeling efficiency>95%,iron particles phagocytosis can be seen within the cells, but this has too much influence inthe proliferation of TSCs.3. LV-GEP can effectively mark TSCs in vitro, mark efficient rises when MOI value goesup. The best MOI value is500. Fluorescence intensity does not decrease when sub culturing invitro decreases and has no obvious influence in osteogenesis, adipogenesis nor chondrogenesis.4. Exogenous TSCs can be implanted in the rat model of Achilles tendon rupturelocation and help with the reparations of tendons damage. Future more, it improves thehistology characteristics without any abnormal differentiation. So the exogenous has thepotential capability to treat acute Achilles tendon rupture.