Alginate, a biocompatible polymer naturally derived from algae, is widely used as a synthetic analogue of the extracellular matrix in tissue engineering. Integrin-binding peptide motifs, including RGD, a derivative of fibronectin, are typically grafted to the alginate polymer through carbodiimide reactions between peptide amines and alginate uronic acids. However, lack of chemo-selectivity of carbodiimide reactions can lead to side reactions that lower peptide bioactivity. To overcome these limitations, we developed an approach for copper-free, strain-promoted azide–alkyne cycloaddition (SPAAC)-mediated conjugation of azide-modified adhesive peptides (azido-cyclo-RGD, Az-cRGD) onto alginate. Successful conjugation of azide-reactive cyclooctynes onto alginates using a heterobifunctional crosslinker was confirmed by azido-coumarin fluorescent assay, NMR, and through click reactions with azide-modified fluorescent probes. Compared to cyclo-RGD peptides directly conjugated to alginate polymers with standard carbodiimide chemistry, Az-cyclo-RGD peptides exhibited higher bioactivity, as demonstrated by cell adhesion and proliferation assays. Finally, Az-cRGD peptides enhanced the effects of recombinant bone morphogenetic proteins on inducing osteogenesis of osteoblasts and bone marrow stromal stem cells in 3D alginate gels. SPAAC-mediated click approaches for peptide–alginate bioconjugation overcome the limitations of previous alginate bioconjugation approaches and potentially expand the range of ligands that can be grafted to alginate polymers for tissue engineering applications.

Mesenchymal stromal cells (MSCs) have been successfully employed in clinical applications. In most studies, autologous MSCs from the bone marrow (bmMSCs) were used, and others employed autologous adipose tissue-derived stromal cells (ADSCs). Recently, clinical feasibility studies provided evidence that MSCs from human term placenta (pMSCs) can be used for homologous therapy facilitating access to regenerative cells in emergency situations, when autologous cells are not available or not suitable. We therefore investigated the expression of MSC stemness marker CD146 and the expression of neuro- and myoregenerative cytokines by human pMSCs after expansion in three different media compliant with good manufacturing protocols (GMP) in comparison to pMSCs expanded in a commercial MSC expansion media. To replace xenobiotic serum in the GMP-compliant media employed in this study, either human serum, human serum plus platelet lysate (PLL), or human plasma plus PLL was used. We report that enrichment of media with PLL accelerates pMSC proliferation but reduces the expression of the stemness marker CD146 significantly, while PLL deprivation enhanced the CD146 expression. In contrast, the reduced expression of CD146 by PLL deprivation was not observed on bmMSCs. The expression of the cytokines investigated was not modulated significantly by PLL. We conclude that accelerated expansion of pMSCs in GMP-compliant media enriched by PLL reduces the expression of stemness marker CD146, but does not influence the expression of neuro- and myoregenerative cytokines.

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