Previous studies have demonstrated that epidermal growth factor (EGF) can promote tissue regeneration effectively. The aim of the present study was to combine a collagen-binding domain (CBD) with native EGF (NAT-EGF) to form a fusion protein, aiming at that it could bind to collagen scaffolds specifically. This approach could restrict the diffusion of EGF despite of extracellular fluids and limit the release of EGF to surrounding tissues. Thus, the retained CBD-EGF would render a continual function during the tissue regeneration. In our study, in vitro bioactivity assay showed that there was no significant difference between CBD-EGF and NAT-EGF to stimulate human fibroblasts to proliferate. In the collagen-binding assay, CBD-EGF exhibited a specific binding ability to collagen compared to NAT-EGF. Finally, in vivo experiment demonstrated that CBD-EGF–loaded collagen membranes uniformly possess a high capability to promote cellularization of scaffolds as compared with that of NAT-EGF. All results aforementioned suggested that collagen loaded with CBD-EGF is a functional biomaterial with potential clinical applications.
PastoreS., MasciaF., MarianiV., GirolomoniG.The epidermal growth factor receptor system in skin repair and inflammation. J Investig Dermatol, 128:1365. 2008.
3.
TripathiR.C., RajaS.C., TripathiB.J.Prospects for epidermal growth factor in the management of corneal disorders. Surv Ophthalmol, 34:457. 1990.
4.
HallF.L., KaiserA., LiuL., ChenZ.H., HuJ., NimniM.E., BeartR.W.Jr., GordonE.M.Design, expression, and renaturation of a lesion-targeted recombinant epidermal growth factor-von Willebrand factor fusion protein: efficacy in an animal model of experimental colitis. Int J Mol Med, 6:635. 2000.
5.
KumarR.K., MaroneseS.E., HassimZ.Cooperative interaction of autocrine and paracrine mitogens for airway epithelial cells. Cell Biol Toxicol, 14:293. 1998.
6.
HardwickeJ., SchmaljohannD., BoyceD., ThomasD.Epidermal growth factor therapy and wound healing—past, present and future perspectives. Surgeon, 6:172. 2008.
7.
FrewS.E., RezaieR., SammutS.M., RayM., DaarA.S., SingerP.A.India's health biotech sector at a crossroads. Nat Biotechnol, 25:403. 2007.
NishiN., MatsushitaO., YuubeK., MiyanakaH., OkabeA., WadaF.Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain. Proc Natl Acad Sci USA, 95:7018. 1998.
16.
de SouzaS.J., BrentaniR.Collagen binding site in collagenase can be determined using the concept of sense-antisense peptide interactions. J Biol Chem, 267:13763. 1992.
17.
ChenB., LinH., ZhaoY., WangB., LiuY., LiuZ., DaiJ.Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand factor propolypeptide. J Biomed Mater Res A, 80:428. 2007.
18.
LinH., ChenB., SunW., ZhaoW., ZhaoY., DaiJ.The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds. Biomaterials, 27:5708. 2006.
19.
Ferrer SolerL., CedanoJ., QuerolE., de LlorensR.Cloning, expression and purification of human epidermal growth factor using different expression systems. J Chromatogr B Anal Technol Biomed Life Sci, 788:113. 2003.
20.
BradfordM.M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72:248. 1976.
21.
SchaggerH., von JagowG.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem, 166:368. 1987.
22.
LembachK.J.Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arginine esterase and by ascorbate. Proc Natl Acad Sci USA, 73:183. 1976.
23.
FinnisM.L., GibsonM.A.Microfibril-associated glycoprotein-1 (MAGP-1) binds to the pepsin-resistant domain of the alpha3(VI) chain of type VI collagen. J Biol Chem, 272:22817. 1997.
24.
NimniM.E.The molecular organization of collagen and its role in determining the biophysical properties of the connective tissues. Biorheology, 17:51. 1980.
25.
MatsushitaO., JungC.M., MinamiJ., KatayamaS., NishiN., OkabeA.A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J Biol Chem, 273:3643. 1998.
26.
OgisoH., IshitaniR., NurekiO., FukaiS., YamanakaM., KimJ.H., SaitoK., SakamotoA., InoueM., ShirouzuM., YokoyamaS.Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell, 110:775. 2002.
27.
PlayfordR.J., MarchbankT., CalnanD.P., CalamJ., RoystonP., BattenJ.J., HansenH.F.Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice. Gastroenterology, 108:92. 1995.
28.
IshikawaT., TeraiH., KitajimaT.Production of a biologically active epidermal growth factor fusion protein with high collagen affinity. J Biochem, 129:627. 2001.
29.
IshikawaT., TeraiH., YamamotoT., HaradaK., KitajimaT.Delivery of a growth factor fusion protein having collagen-binding activity to wound tissues. Artif Organs, 27:147. 2003.
30.
LeeJ.H., AhnS.H., LeeE.M., JeongS.H., KimY.O., LeeS.J., KongI.S.The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen. FEBS Lett, 579:2507. 2005.
31.
KimD.G., MinM.K., AhnS.C., KimJ.K., KongI.S.Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of Vibrio mimicus metalloprotease. Biotechnol Lett, 31:259. 2009.
