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Thymosin-β4, and Human Vitronectin peptides Grafted to Collagen Tune Adhesion or VEGF Gene Expression in Human Cell Lines
Collagen is the most abundant protein in the human body, found in the bones, muscles, skin, and tendons. It provides structural support to the extracellular space of connective tissues. Due to its rigidity and resistance to stretching, it is the perfect matrix for skin, tendons, bones, and ligaments.
Researchers wanted to test the modulation of adhesiveness strength and specificity of collagen scaffolds through the grafting of adhesive peptides. They thought that this may improve both cell adhesion and migration, favoring the tissue regenerative process. However, to date, no such study has been performed.
More specifically, in the study, researchers examined thymosin-β4 (Tβ4P) and Human Vitronectin (HVP) (seen in the image to the right) derived peptides grafted to collagen by thiolene Michael addition in order to improve collagen bioactivity for regenerative medicine approaches.
Tβ4P and HVP are known to exert proangiogenic and proadhesive activity respectively, and HVP is involved in osteogenesis promotion. The ability of these peptides to increase collagen cell adhesion and angiogenesis properties is assessed on human cell lines. See image below showing the peptides grafting strategy to collagen coatings.
The results appeared to be very promising. In particular, HVP-grafted collagen increased human osteoblast adhesion and cell proliferation: after 24 h, both adhesion and proliferation roughly showed a 4-fold increase, if compared to pristine collagen. Tβ4P-grafted collagen promotes Vascular Endothelial Growth Factor (VEGF) gene expression in human vascular cell lines by more than 7 times. See Figure 1 and Figure 2 below.
Figure 1. HVP-Collagen drives adhesion and proliferation of h-osteoblasts. Adhesion was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test following 2 or 24 h of culture. Cell proliferation was evaluated by flow cytometry analysis in CFSE (carboxyfluorescein succinimidyl ester) loaded cells. * Denotes P<0.02 vs pristine collagen at the same culture time. Data are reported as mean +/- standard error of two independent experiments, each performed in triplicate. Statistical analysis was performed using the ONE-way ANOVA test followed by Bonferroni’s multi comparison test.
Figure 2. Tβ4P-Collagen induces expression of VEGF in Human Dermal Lymphatic Endothelial Cells (HDLEC). Specific VEGF mRNA transcript levels were assessed
in HDLEC cultured for 48 h. Data were calculated as fold expression over cells cultured on plastic supports. Proliferation of HDLEC cultured for 24 h on Tβ4P- Collagen was evaluated by flow cytometry analysis in CFSE loaded cells. *Denotes P<0.02 vs pristine collagen at the same culture time. Data are reported as mean +/- standard error of two independent experiments, each performed in duplicate. Statistical analysis was performed using the ONE-way ANOVA test followed by Bonferroni’s multi comparison test.
These results suggest that HVP-grafted collagen may be an interesting biomaterial for bone tissue regeneration, while Tβ4P-grafted collagen is useful for angiogenesis promotion. More studies about peptide concentrations, their topological localization on the matrix surfaces, enzymatic degradation by cellular enzymes, need to be taken into account in future studies.
You can read the full article here.
Roberto Guizzardi, Annj Zamuner, Paola Brun, Monica Dettin, Antonino Natalello, Laura Cipolla, Thymosin-β4, and Human Vitronectin peptides Grafted to Collagen Tune Adhesion or VEGF Gene Expression in Human Cell Lines, Chemistry Europe (2021), doi: https://doi.org/10.1002/slct.202102757
