In Vivo Effects Of Adipose Derived Mesenchymal Stem Cells Reseeding On Acellular Bovine Pericardium

【Background】For the past few years, as some difficulties, erosion, exposure, dyspareunia,constraction，fistula, advent in pelvic floor reconstructive surgery, tissue engineering hadbeen gained more and more attention in female pelvic floor reconstructive surgery research,becoming a new and expanding field in female pelvic floor reconstructive surgery.However, how long they survive, where they distribution, and what they will differentiateto in living animals remain unknown. In this study， we constructed one kind ofadipose-derived stem cells co-combined with bovine pericardium graft to transplant innude mice, and then used non-invasive bioluminescence imaging, histology andimmunohistochemistry to track in real time the survival time, distribution, differentiationof human adipose-derived stem cells, investigating the feasibility of humanadipose-derived stem cells reseeded into acellular bovine pericardium grafts in pelvic floorreconstructive surgery.【Methods】1. Preparation in vitro of bioactive materials1.1human ASCs isolation and culture, identification, labeling and selecting:(1) Human ASCs were isolated from a35–year-old female adipose tissue harvestedfrom cosmetic subdermal liposuction with patient consent by utilizing enzymatic digestion,adherence. Human ASCs were observed under microscope to record their morphology andproliferating characteristics. The phenotype of hASCs was detected by flow cytometry todemonstrate their ability of multi-directional differentiation.(2) Lentiviral vectors containing enhanced green fluorescent protein (eGFP) gene andLuciferase (Luc) gene transfected with the third-passage cells. The expression of eGFPwas then observed by fluorescence microscope after transfection.(3) The hASCs infected with recombinant lentivirus were selected and purified by puromycin. The transduction efficiency of eGFP levels were evaluated by flowcytometry.1.2Construction of the bioactive materials(1) EGFP·Luc-hASCs was co-cultured with the ABP (provided by ShanghaiCingular Biotech Corporation) in vitro.(2) The cell viability of eGFP·Luc-hASCs was evaluated by the Cell-Counting Kit(CCK)-8assays. EGFP·Luc-hASCs at passage8were seeded on acelluar bovinepericardium in vitro, and then CCK-8solution reagent was added at each time point to plotthe curve of cell viability.1.3Luciferase assays(1) In vitro, eGFP·Luc-hASCs of different numbers (10,100,500,1000,5000,10000)/100μl at passage6were seeded onto black, clear bottom96well platesrespectively, and then added D-luciferin solution. A highly sensitive, cooled chargecoupled device (CCD) camera was used to detect the bioluminescent signals.Quantification of signals was controlled by the acquisition and analysis software LivingImage.(2) In vivo, eGFP·Luc-hASCs of different numbers (1×106/100μl,1×105/100μl,1×104/100μl,1×103/100μl,100/100μl）were grafted in subcutaneous space ineach dorsum of nude mice. The animals were intraperitoneal injected with luciferin andthen were directly imaged by CCD. Quantification of signals was evaluated by theacquisition and analysis software Living Image.2. In vivo effects of bioactive materials experiment2.1Bioactive materials transplanting in vivo:Thirty athymic nude mice (BALB/C-nu/nu) were randomized into three groups of teneach. The eGFP·Luc-hASCs reseeded graft was implanted within the subcutaneous pocketof ten mice at the same time (LOCAL-INJE group,n=10). Another ten mice was implantedwith graft which co-cultured with eGFP· Luc-hASCs in vitro prior to implanting(CO-CULTURE group,n=10). The last ten mice were implanted with ABP only as controls(BLANK group,n=10). 2.2Bioactive materials analysis(1) Bioluminescence imaging in vivo and analysisBioluminescence imaging of mice was performed in one hour, one week,2weeks,3weeks,4weeks,6weeks,8weeks,12weeks after implantation respectively to detect thebehavior of cells in vivo.(2) Histological and immunohistochemistry analysisAfter12weeks post-implantation, the samples of implants, hearts, livers, lungs wereharvested for histological analysis. One histological section from the postoperative frozensections was used for DAPI staining to detect eGFP-positive cell. H﹠E staining, Massontrichrome, Immunohistochemical staining were to evaluate effects of bioactive materials.【Results】1. The morphologies of hASCs were spindle, radial shaped or a swirling distribution.hASCs labeled with eGFP-Luc gene had a high eGFP and Luc expression level in vitro.The eGFP-Luc lentivirus vectors infecting rate of hASCs were up to91.12%by flowcytometry.2. EGFP·Luc-hASC showed well adherence to ABP. There were no significantdifferences between the viability of hASCs and that of eGFP-Luc labeled hASCs in theCCK-8assay.3. In vitro and in vivo BLI imaging of serial dilution of eGFP·Luc-hASCs indicatedthat the number of eGFP·Luc-hASCs was linearly correlated with light production,indicating that the BLI signals could be used to quantitatively track labeled cells.4. BLI was performed one day post-implantation and thereafter at the indicated times.The results showed that both of eGFP-Luc hASCs in two groups remained viable in vivoup to12weeks. At the following three weeks, the cells number in two groups bothdecreased rapidly. During the rest six weeks observation, the number of surviving cellsremained stable until the end of the experiment. The majority of implanted eGFP-LuchASCs existed in the location of inoculating. No significant signal was detected in otherorgans such as lung, liver, heart, or brain.5. EGFP-positive ASCs were detected at subcutaneous layer in the DAPI staining offrozen sections, confirming that implanted cells survived. No eGFP-positive cell wasdetected in other organs; the results of H&E staining showed that both LOCAL-INJE andCO-CULTURE group displayed numerous small vessels and cells ingrowths. Little vesselsand cells ingrowths were displayed in BLANK group; Masson trichrome staining revealedthat more collagen was formed in the reseeded group compared to the BLANK group; immunohistochemical staining showed that there was a significantly healthymyofibroblasts or smooth muscle cells in LOCAL-INJE group. However, there was very alittle smooth muscle in CO-CULTURE and blank group.【Conclusions】After bioactive materials was transplanted to mice, human adipose-derived stem cellscould survive in vivo long-termly, which could slow down the degradation rate of ABP,promote the cells ingrowths, vascularization, muscular regeneration. Bioactive materialswhich hASCs reseeded on ABP during surgery may be a promising approach for thetreatment of pelvic floor reconstruction.