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The mechanical strength, thermal and structural stability, resistance to biodegradation and cell viability of this scaffold was investigated. The results of the Coll\u2010<jats:sc>D<\/jats:sc>\u2010Lys\u2010EDC\/NHS scaffold also indicate an increase in the tensile strength (<jats:italic>T<\/jats:italic><jats:sub>S<\/jats:sub>), percentage of elongation (% <jats:italic>E<\/jats:italic>), denaturation temperature (<jats:italic>T<\/jats:italic><jats:sub>d<\/jats:sub>), and decrease the decomposition rate. Scanning electron microscopic (SEM) and atomic force microscopic (AFM) analyses revealed a well ordered with properly oriented and well\u2010aligned structure of scaffold. The <jats:sc>D<\/jats:sc>\u2010Lys stabilizes the scaffold against degradation by collagenase than <jats:sc>L<\/jats:sc>\u2010Lys. The cell assay showed more than 98 \u00b1 2% fibroblast viability (NIH 3T3) after 72 h of culture Coll\u2010<jats:sc>D<\/jats:sc>\u2010Lys\u2010scaffold when compared with native Coll and Coll\u2010<jats:sc>L<\/jats:sc>\u2010Lys\u2010scaffold. The proteolytic machinery is not well equipped to deal with Coll\u2010<jats:sc>D<\/jats:sc>\u2010Lys\u2010scaffold than Coll\u2010<jats:sc>L<\/jats:sc>\u2010Lys\u2010scaffold. Incorporating <jats:sc>D<\/jats:sc>\u2010Lys in scaffold design has the potential to improve existing collagen stability and create new topologies inaccessible to homochiral molecules. This method may assist in the functionalization of the scaffold for regenerative applications. \u00a9 2012 Wiley Periodicals, Inc. 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