Bladder Regeneration By Collagen Scaffolds With Collagen-binding Human Basic Fibroblast Growth Factor

Various injuries, such as cancer, trauma, infection, inflammation, or congenital abnormalities, may result in compromised bladder structure and compliance, which requires reconstruction. Currently, the major surgical solution is enterocystoplasty by vascularized bowel segments. However, multiple significant complications of this method greatly hamper its clinical application. It is important to improve the current method or find a better alternative for bladder regeneration.In this study, we constructed a targeted-repair functional biomaterial, A collagen-binding domain (CBD) was added to the N-terminal of native bFGF. With the CBD, bFGF could bind to the collagen scaffold specially. The CBD-bFGF/collagen is remarkable for its weak antigenicity, excellent biodegradability and targeting delivery of bFGF, On the one hand the system can be targeting and sustaining delivery of regeneration of signaling molecules, while exogenous collagen scaffold provides a three-dimensional space for cell proliferation and tissue regeneration.This study including the following three parts:Part I: CBD-bFGF, NAT-bFGF protein expression, purification and identification.In this part of the experiment, through engineering technology, we designed expression sequence for CBD-bFGF, NAT-bFGF protein, using pET28a vector for protein expression, Ni column for affinity purification. After protein expression, the bands between the 19kD and 21kD were obvious observed on NAT-bFGF recombinant protein and CBD-bFGF recombinant protein by 15% SDS-PAGE. The reason why the CBD-bFGF and NAT-bFGF protein were at two different bands was that a CBD was in the CBD-bFGF, so that CBD-bFGF protein was larger than NAT-bFGF protein. In order to acquire more accurate results, we also did a western-blotting analysis and two clear bands were seen at the targeted location. Part II: CBD-bFGF protein, NAT-bFGF protein biological activity and CBD-bFGF/ Collagen and NAT-bFGF/Collagen repair material preparation.We chose MTT analysis of proliferation of human fibroblasts experiments to detect the biological activity of the expressed protein. CBD-bFGF and NAT-bFGF stimulated proliferation of fibroblasts for three days, and then added MTT, after DMSO dissolved, OD492 value was measured. The results indicated that the proliferation of fibroblasts showed obvious dose-dependent effect. With the protein concentration increased, cell proliferation rate also accelerated. There was no significant proliferation difference between CBD-bFGF and NAT-bFGF group. Statistics results showed no significant differences between the two groups at each concentration of OD values, indicating that CBD-bFGF was not only biologically active, but also the activity of were similar with NAT-bFGF.After detection of biological activity of CBD-bFGF and NAT-bFGF, collagen binding capacity was also analyzed to confirm whether the CBD-bFGF could bind effectively with the collagen. CBD-bFGF and NAT-bFGF were added to collagen-coated 96-well plate for combination, and then ELISA was used to detect the binding capacity. From the results, we found at each indicative point, CBD- bFGF retained on the collagen was more than that of NAT-bFGF detected by the absorb value at 405 nm. This result reflected that the CBD- bFGF had a specific binding ability to collagen and the collagen-binding capacity greatly increased.The collagen scaffolds were loaded with equivalent NAT-bFGF or CBD-bFGF and soaked in PBS. At day 0 to 14, we analyed the NAT-bFGF and CBD-bFGF retained on the collagen scaffolds by ELISA assay to confirm the release rate. Results showed that CBD-bFGF retained on the collagen scaffolds was significantly higher than NAT-bFGF from day 1 to 7. The CBD-bFGF release rate from collagen was slower than NAT-bFGF, indicating CBD-bFGF with a better ability to control release from the collagen scaffolds.Collagen-coated 48-well plates were incubated with NAT-bFGF or CBD-bFGF for 1 h, and then washed by PBS for 4 times. Fibroblasts were seeded in these wells. After 72 h incubation, the proliferation of fibroblasts was detected by MTT assay. In different protein concentration, cell proliferation rate in CBD-bFGF group was significantly higher than NAT-bFGF group. Statistics results showed significant differences between the two groups at OD values, indicating that CBD-bFGF could retain on the collagen scaffold with a higher biological activity.Part III: CBD-bFGF/Collagen and NAT-bFGF/Collagen repair materials on rat bladder injury mode.We conducted in vivo functional experiments. We chose rat bladder reconstruction model to further evaluate the repair effects of CBD-bFGF/Collagen system. Four groups: CBD-bFGF/Collagen, NAT-bFGF/Collagen, PBS/Collagen and Sham-operated group. 30d and 90 d was the point of observation. The results showed that CBD-bFGF/Collagen group could promote more vascularization and ingrowth of smooth muscle cells than those of PBS/Collagen group and NAT-bFGF/Collagen group. Urodynamic examinations revealed well-accommodated bladder tissue with volume capacity and compliance similar with Sham-operated group. The results showed that CBD-bFGF/Collagen targeting delivery system played an important role at the injured site.Based on the above, by genetic engineering methods, a collagen-binding domain (CBD) which could bind to the collagen scaffold specially was added to the N-terminal of native bFGF. Results showed that CBD-bFGF, while maintaining the biological activity, had a specific binding ability to collagen. In the rat bladder reconstruction model, CBD-bFGF/Collagen targeted repair system promoted better bladder tissue regeneration and functional recovery, showing the enormous potential of CBD-bFGF/Collagen targeted repair material in bladder reconstruction. This study explored using tissue engineering methods for bladder substitute materials to provide new technologies and methods for the reconstruction with broad research values and potential clinical applications.