Table 1.
Primers for Real-Time Polymerase Chain Reaction
Fig 1.
Multipotent differentiation of hASCs in vitro.
(A) Oil Red O staining detected red-colored oil droplets in adipogenic differentiation of hASCs; (B) Alizarin Red detected calcium mineralization in osteogenic differentiation of hASCs; (C and D) For chondrogenic differentiation, histological and H&E staining results showed that cartilage lacunae were formed and expressed chondrocyte gene marker, collagen II.
Fig 2.
Physical characteristics of PBLG microspheres.
(A, B) SEM examination of the whole porous PBLG microcarriers. (C, D) Cross-sectional view of porous PBLG microcarriers. (E) Porosity and pore diameters of PBLG microcarriers (n = 3). (F) SEM examination of injected porous PBLG microcarriers.
Fig 3.
Biological characteristics of hASCs growing within porous PBLG microspheres.
(A) Confocal images of the live (green)/dead (red) assay for the hASCs growing in microcarriers 48 h post-seeding. (B) Confocal laser microscopy observation of Hoechst33258-stained hASCs growing within the porous PBLG microcarriers at 6, 12, 24, and 48 h. (C) Confocal laser microscopy observation of Hoechst33258-stained hASCs at the indicated depth in the microsphere after cell seeding for 48 h. (D) hASC proliferation within PBLG microspheres maintained in adipogenic medium (AM) or growth medium (GM) for 14 d (n = 3). *P < 0.05; **P < 0.01.
Fig 4.
Adipogenic differentiation of hASCs within PBLG microspheres.
(A) Real-time PCR detection of the expression of adipogenic genes, including aP2, C/EBP α, LPL, and PPAR γ, at the indicated time points (n = 3). (B) GPDH enzyme activity of the hASCs growing within the PBLG microspheres maintained in adipogenic medium (AM) or growth medium (GM) (n = 3). *P < 0.05; **P < 0.01.
Fig 5.
Construction, harvest and analysis of the neo-generated tissues.
(A) Subcutaneous injection of hASC/PBLG microsphere complex and harvest of neo-generated tissue after 8 weeks. (B) Weight and volume analysis of the neo-generated tissues (n = 3) (P > 0.05). n.s. = no statistical significance.
Fig 6.
H&E, Masson’s trichrome, Oil Red O staining, and scanning electron microscope (SEM) examination of the neo-generated tissue in the three groups at 4 and 8 weeks post-injection.
(A) PBLG group: injection of PBLG microsphere alone; (B) ASC/PBLG group: injection of non-induced hASC/PBLG complex; and (C) Adi-ASC/PBLG group (4 weeks): injection of adipogenic-induced hASC/PBLG microsphere complex.
Fig 7.
Histological analysis of normal human adipose and neo-generated tissues harvested from the Adi-ASC/PBLG group 8 weeks post-injection.
(A) H&E and Masson’s trichrome staining of normal human adipose and neo-generated fat tissues; (B) Comparative analysis of the number of vessel lumina and luminal diameters between normal human adipose and neo-generated tissues (n = 3) (P > 0.05). n.s. = no statistical significance.
Fig 8.
GFP-labeled hASC detection in the engineered adipose tissues by confocal laser microscopy observation at 4 and 8 weeks post-injection.
Cell nuclei were counterstained with Hoechst 33258 dye.
Fig 9.
Biochemical analysis of neo-generated tissue from different groups after 8 weeks of treatment.
(A) qRT-PCR analysis of adipogenic gene expression from the harvested tissue (n = 3). (B) GPDH enzyme activity of the neo-generated tissues harvested from the three groups at 4 and 8 weeks post-injection (n = 3). (C) Hydroxyproline content in the neo-generated tissue. Human adipose was used as the positive control (n = 3). *P < 0.05; **P < 0.01.