Plastic Surgery Research Council
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Serum-free Ex Vivo Quality and Quantity Cultured Endothelial Progenitor Cells Improve the Fat Graft Vascularization and Survival
Maxim Geeroms, MD1,2, Moustapha Hamdi, MD, PhD2, Hiroshi Mizuno, MD, PhD1, Rica Tanaka, MD, PhD1.
1Plastic and Reconstructive Surgery Department, Juntendo University School of Medicine, Tokyo, Japan, 2Plastic and Reconstructive Surgery Department, Brussels University Hospital (UZ Brussel), Vrije Universiteit Brussel, Brussels, Belgium.

PURPOSE: Research has not yet demonstrated a reliable solution to overcome the postoperative volume loss in fat grafting. The establishment of an early blood supply is important to prevent tissue necrosis for higher fat engraftment. We have recently developed an ex vivo expansion system named Quality and Quantity control culture system (QQc) which strongly stimulates the vasculogenic potential of endothelial progenitor cells (EPCs) and multiplies their number. We hypothesized that the addition of EPCs, enhanced by the QQ culture, to freshly harvested adipose tissue aids in the establishment of a blood vessel network which leads to better graft survival.
METHODS: C-kit+ Sca-1+ lineage-negative (KSL) cells were isolated as EPC-precursors from C57BL/6 mice and cultured for 7 days in QQc. Adipose tissue, harvested from C57BL/6 mice and weighing 0.25g per graft, was transplanted with 2x104 QQc-cultured KSL-cells (group 1, n=18) or with 2x104 non-cultured KSL-cells (group 2, n=10), while controls received the adipose tissue alone (group 3, n=18). 5 and 10 weeks later, the grafts were explanted and graft survival was evaluated by weight maintenance. Histological and immunohistochemical assessment of the grafts was performed after 5 weeks. The expression of the angiogenic marker PDGF-B and the adipocyte-specific markers adiponectin and FABP4 in the grafted tissue was analyzed by qRT-PCR after 5 weeks and compared to group 3.
RESULTS: Group 1 demonstrated the highest graft survival after 5 weeks (58.2±23.8%) compared to group 2 (51.1±21.3%, p>0.05) and group 3 (40.3±19.7%, p<0.05). The graft weight remained stable between 5 and 10 weeks after grafting. CD31 immunohistochemistry confirmed a higher vessel density in group 1 (35.0±7.4/mm²) in comparison with groups 2 (28.4±5.5/mm², p<0.01) and 3 (20.8±6.4/mm², p<0.0001). In addition, the lowest percentage of fibrotic tissue was found in group 1 (group 1: 13.8±10.8%; group 2: 20.9±10.1%; group 3: 21.9±11.4%, p<0.01) and grafts treated with QQc-cultured KSL-cells showed less CD68-positive local inflammation units (31.4±21.9/mm² vs 40.2±26.7/mm² vs 42.8±33.5/mm², p<0.05). Moreover, we observed a significant upregulation of PDGF-B (1.7-fold change, p<0.01), adiponectin (1.9-fold change, p<0.05) and FABP4 (1.9-fold change, p<0.05) in group 1.
CONCLUSIONS: The addition of QQc-cultured EPC to murine fat grafts improves their survival by stimulating neovascularization. This is associated with a higher vessel density, less fibrosis, less inflammatory infiltrates and an increase in adipocyte-specific markers. Since the QQc culture greatly increases the EPC number and their vasculogenic potential, an optimal result can be achieved from an initially small number of cells. In the future, QQc-cultured EPC may lead to a more stable postoperative result in human fat grafting.


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