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The successful isolation of adipose-derived mesenchymal stem cells (ADSCs) from the arthroscopically harvested infrapatellar fat pad (IFP) would provide orthopaedic surgeons with an autologous solution for regenerative procedures.To demonstrate the quantity and viability of the mesenchymal stem cell population arthroscopically harvested from the IFP as well as the surrounding synovium.Descriptive laboratory study.The posterior border of the IFP, including the surrounding synovial tissue, was harvested arthroscopically from patients undergoing anterior cruciate ligament reconstruction. Tissue was then collected in an AquaVage adipose canister, followed by fat fractionization using syringe emulsification and concentration with an AdiPrep device. In the laboratory, the layers of tissue were separated and then digested with 0.3% type I collagenase. The pelleted stromal vascular fraction (SVF) cells were then immediately analyzed for viability, mesenchymal cell surface markers by fluorescence-activated cell sorting, and clonogenic capacity. After culture expansion, the metabolic activity of the ADSCs was assessed by an AlamarBlue assay, and the multilineage differentiation capability was tested. The transition of surface antigens from the SVF toward expanded ADSCs at passage 2 was further evaluated.SVF cells were successfully harvested with a mean yield of 4.86 ± 2.64 × 105 cells/g of tissue and a mean viability of 69.03% ± 10.75%, with ages ranging from 17 to 52 years (mean, 35.14 ± 13.70 years; n = 7). The cultured ADSCs composed a mean 5.85% ± 5.89% of SVF cells with a mean yield of 0.33 ± 0.42 × 105 cells/g of tissue. The nonhematopoietic cells (CD45−) displayed the following surface antigens as a percentage of the viable population: CD44+ (52.21% ± 4.50%), CD73+CD90+CD105+ (19.20% ± 17.04%), and CD44+CD73+CD90+CD105+ (15.32% ± 15.23%). There was also a significant increase in the expression of ADSC markers CD73 (96.97% ± 1.72%; P < .01), CD10 (84.47% ± 15.46%; P < .05), and CD166 (11.63% ± 7.84%; P < .005) starting at passage 2 compared with freshly harvested SVF cells. The clonogenic efficiency of SVF cells was determined at a mean 3.21% ± 1.52% for layer 1 and 1.51% ± 0.55% for layer 2. Differentiation into cartilage, fat, and bone tissue was demonstrated by tissue-specific staining and quantitative polymerase chain reaction.SVF cells from the IFP and adjacent synovial tissue were successfully harvested using an arthroscopic technique and produced ADSCs with surface markers that meet criteria for defined mesenchymal stem cells.An autologous source of stem cells can now be harvested using a simple arthroscopic technique that will allow orthopaedic surgeons easier access to progenitor cells for regenerative procedures.