Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion.
Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.
National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. gopinath25@gmail.com
Gopinath
G
Damodaran
Russell
R
Collighan
Martin
M
Griffin
Harshad
H
Navsaria
Abhay
A
Pandit
D016475:3T3 Cells; D000042:Absorption; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D040761:Biomimetic Materials / Q000737:chemistry; D002448:Cell Adhesion / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D003094:Collagen / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D005109:Extracellular Matrix / Q000737:chemistry; D007797:Laminin / Q000737:chemistry*; D008422:Materials Testing; D051379:Mice; D010316:Particle Size; D016062:Porosity; D013499:Surface Properties; D023822:Tissue Engineering / Q000379:methods*; D011503:Transglutaminases / Q000737:chemistry*
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D007797:Laminin; C095567:laminin alpha 3; D003094:Collagen; D011503:Transglutaminases