[Comparison between kinds of myofascial flap encapsulating adipose-derived stromal cells carrier complex in terms of adipogenic efficacy in vivo].

OBJECTIVE: To compare two kinds of myofascial flap encapsulating adipose-derived stromal cells (ADSCs) in adipogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adipose tissue.

METHODS: ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3- 4-months-old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 microg/mL) was applied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to those cells 2 weeks after being induced towards adipocyte, alizarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 x 10(7) ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm x 10 mm x 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was applied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothelium.

RESULTS: The nucleus of ADSCs positive for BrdU labeling showed green fluorescence under fluorescence microscope, with the positive labeling ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red lipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibre encapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 +/- 0.017 3) g and (0.095 3 +/- 0.012 7) g, respectively, indicating there was a significant difference between two groups (P < 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 +/- 4.5 and 19.3 +/- 2.6, respectively, indicating there was a significant difference between two groups (P < 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adipocytes and partial capillary endothelium in groups A and B presented green fluorescence.

CONCLUSION: ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adipogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.