Basic fibroblast growth factor delivery enhances adrenal cortical cellular regeneration

Abstract

The effective delivery of angiogenic factors is a useful strategy for the engineering of vascularized tissues. When adrenal cortical cells were implanted in mice under the renal capsule, the size of the implant was reduced to about 100 microm in thickness after 8 weeks. Either low (approximately 2 microg) levels of basic fibroblast growth factor (bFGF) or high (approximately12 microg) levels of bFGF were encapsulated into poly-lactic-co-glycolic acid microspheres, and these bFGF-encapsulated microspheres were coimplanted with adrenal cortical cells. After 56 days, the implants with low and high levels of bFGF weighed five and eight times more, respectively, than the implants without bFGF delivery. The implants with bFGF-encapsulated microspheres also contained significantly more cells than the implants without bFGF delivery. The levels of adrenal cortical gene expression were not significantly changed with bFGF delivery. The implants with high levels of bFGF also had a more uniform distribution of anti-CD31 immunofluorescence. Based on the increased number of cells that expressed adrenal cortical genes, the delivery of bFGF enhanced adrenal cortical cellular regeneration, possibly through an angiogenic response.

FIG. 1.

Western blot analysis of bFGF extracted from PLGA microspheres. Lanes 1, 2, and 3: 100, 400, and 600 ng of bFGF, respectively; lanes 4, 5, and 6: bFGF extracted from the control, the low, and the high bFGF groups, respectively.

FIG. 2.

Weights of retrieved implants at different time points. Implants were retrieved on days 10, 28, and 56. The weight of the high bFGF group was compared to that of the control and low bFGF groups. The weight of the low bFGF group was compared to that of the control group. *p < 0.05 as compared to the control group, and ^#p < 0.05 as compared to the low bFGF group. Error bar represents standard deviation of measurements of retrieved implants from three animals.

FIG. 3.

Histological appearance of tissues formed from the implanted adrenal cortical cells and bFGF delivery. Hematoxylin/eosin stain, 100×. (A), (B), and (C) were retrieved on day 10; (D), (E), and (F) were retrieved on day 28; (G), (H), and (I) were retrieved on day 56. (A), (D), and (G) are from the control group; (B), (E), and (H) are from the low bFGF group; (C), (F), and (I) are from the high bFGF group. Scale bar, 100 μm. k, kidney. The micrographs are representative of results from three experiments. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Average cell numbers within the retrieved implants. Implants were retrieved on days 10, 28, and 56. The cell number of the high bFGF group was compared to that of the control and low bFGF groups. The cell number of the low bFGF group was compared to that of the control group. *p < 0.05 as compared to the control group, and ^#p < 0.05 as compared to the low bFGF group. Error bar represents standard deviation of measurements of retrieved implants from three animals.

FIG. 5.

Immunofluorescence of anti-CD31 staining. (A), (B), and (C) were retrieved on day 10; (D), (E), and (F) were retrieved on day 28; (G), (H), and (I) were retrieved on day 56. (A), (D), and (G) are from the control group; (B), (E), and (H) are from the low bFGF group; (C), (F), and (I) are from the high bFGF group. Arrows indicate location of the implants. k, kidney. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Relative intensity of anti-CD31 staining per area of implants. Images of implants retrieved on days 10, 28, and 56 were analyzed as described in Materials and Methods section. Relative intensity per pixel of each group was calculated by normalizing to that of the control group on day 10. The values are means ± standard deviation of measurements from two experiments.

FIG. 7.

Sf1 gene expression in the control, low bFGF, and high bFGF group. Expression level was normalized to pooled neonatal adrenal glands. The gene expression level of implants retrieved at later time points was compared to that of earlier time points. *p < 0.05 as compared to the day 0; ^#p < 0.05 as compared to day 10; ⁺p < 0.05 as compared to day 28. Error bar represents standard deviation of measurements of retrieved implants from three animals.

FIG. 8.

Relative gene expression of adrenal cortical markers in retrieved implants. Implants were retrieved on days 10, 28, and 56. (A) Relative gene expression levels of cells before implantation. (B–D) Relative gene expression levels of implants on days 10, 28, and 56, respectively. The relative expression level of each adrenal-specific gene was normalized to that of the control group from the same time point. The gene expression of the high bFGF group was compared to that of the control and low bFGF groups. The gene expression of the low bFGF group was compared to that of the control group. *p < 0.05 as compared to the control group, and ^#p < 0.05 as compared to the low bFGF group. Error bar represents standard deviation of measurements of retrieved implants from three animals.