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. 2010 Feb;31(5):878-85.
doi: 10.1016/j.biomaterials.2009.10.007. Epub 2009 Oct 23.

Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering

Yonggang Pang  1 , Xiaoli Wang, Areck A Ucuzian, Eric M Brey, Wilson H Burgess, Kathryn J Jones, Thomas D Alexander, Howard P Greisler
Affiliations
Free PMC article

Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering

Yonggang Pang et al. Biomaterials. 2010 Feb.
Free PMC article

Abstract

We investigated the delivery of R136K-CBD (a collagen-binding mutant chimera of fibroblast growth factor-1) with a type I collagen scaffold as the delivery vehicle to smooth muscle cells (SMCs) for vascular tissue engineering. The binding affinity of R136K-CBD to 3-D collagen scaffolds was investigated both in the presence and absence of cells and/or salts. 2-D and 3-D visualization of delivery of R136K-CBD into SMCs were accomplished by combined fluorescent and reflection confocal microscopy. The mitogenic effect of collagen-immobilized R136K-CBD on SMCs in 3-D collagen was studied by Cyquant assay at different time intervals. In the group devoid of salt and cells, no detectable release of R136K-CBD into overlying culture media was found, compared with burst-and-continuous release of R136K and FGF-1 over a 14-day period in all other groups. The release rate of R136K-CBD was 1.7 and 1.6-fold less than R-136K and FGF-1 when media was supplemented with 2m salt (P<0.0001), and 2.6 and 2.5-fold less in cell-populated collagen hydrogels (P<0.0001), respectively. R136K-CBD showed essentially uniform binding to collagen and its distribution was dependent on that of the collagen scaffold. Internalization of R136K-CBD into SMCs was documented by confocal microscopy. 3-D local delivery of collagen-immobilized R136K-CBD increased the proliferation of SMCs in the collagen matrix to significantly greater levels and for a significantly greater duration than R136K or FGF-1, with 2.0 and 2.1-fold more mitogenicity than R136K and FGF-1 respectively (P<0.0001) at day 7. The results suggest that our collagen-binding fusion protein is an effective strategy for growth factor delivery for vascular tissue engineering.

Figures

Fig. 1
Fig. 1
The cumulative release curve of R136K-CBD, R136K and FGF-1. R136K-CBD showed undetectable release for up to 14 days and is significantly less than release of R136K and FGF-1(P<0.0001). Both R136K and FGF-1 demonstrated burst release during the first two days and continuous release in the following days.
Fig. 2
Fig. 2
R136K-CBD showed significantly lower percentage release from collagen hydrogel when supplemented with 2M salt in the media compared with R136K and FGF-1. Asterisk (*) indicates statistical difference at P<0.0001.
Fig. 3
Fig. 3
After 16-hour culture, R136K-CBD showed significantly lower percentage release in collagen hydrogels populated with SMCs compared with R136K and FGF-1 . R136K-CBD showed 2.6 and 2.5 fold less release than R136K and FGF-1, respectively. Asterisk (*) indicates statistical difference at P<0.0001.
Fig. 4
Fig. 4
2-D and 3-D view of Alexa Fluor 488 labeled R136K-CBD inside the collagen scaffold. R136K-CBD and collagen fibers are visualized by florescent and reflection confocal microscopy, respectively.
Fig. 5
Fig. 5
The column chart shows the distribution of Alexa Fluor 488 labeled R136K-CBD within the collagen scaffold. The height of the column represents the mean percentage fluorescence intensity of each stack image. Five stack images were randomly selected from each scaffold and four collagen scaffolds were analyzed. There is no significant difference between any two stack images (n=20, P>0.05).
Fig. 6
Fig. 6
R136K-CBD localized to the cytoplasm and perinuclear area of the SMC. A) An orthogonal view of Alexa fluor 488 labeled R136K-CBD. Middle square box is a focal plane within the SMC in Z axis and the two rectangular boxes on the top and right are the side views of the whole stack image. The existence of R136K-CBD in the middle layer in Z axis proved the internalization. B is the DIC image and C is the overlay.
Fig.7
Fig.7
A is the channel of Alexa fluor 488 (green) labeled R136K-CBD and B is the overlay of R136K-CBD and PKH26 (red) labeled SMC, both visualized with fluorescent confocal microscopy. A and B are the focal planes within the cell in Z axis. C is the 3-D view of R136K-CBD internalizing into the SMC in collagen. Collagen fibers were visualized with reflection confocal microscopy (blue). The green dots inside SMCs in B and the triple-color overlaid white dots inside the SMC demonstrate the internalization of R136K-CBD.
Fig. 8
Fig. 8
Proliferation in response to R136K-CBD continued for the full 7 day period of times compared to the decrease in proliferation of R136K and FGF-1 stimulated groups at day 7. By day 7, proliferation in response to R136K-CBD was 5.5, 2.0 and 2.1 fold greater than R136K, FGF-1 and control groups respectively. Asterisk (*) indicates statistical differences at P<0.0001.
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