evaluation-keratinocyte-proliferation-on-two-three-dimensional-type-i

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Nippi Research Institute of Biomatrix

In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-β1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways.

Type I collagen induces mesenchymal cell differentiation into myofibroblasts through YAP-induced TGF-β1 activation.

Collagen-derived hydroxyproline (Hyp)-containing oligopeptides, known to have various physiological functions, are detected in blood at markedly higher concentrations after oral ingestion of collagen hydrolysate. Monitoring the absorption and metabolism of the bioactive peptides is essential to investigate the beneficial effects of collagen hydrolysate. We previously developed an internal standard mixture by sequential protease digestion of stable isotope-labeled collagen, which enabled highly accurate quantitation of collagen-derived oligopeptides by liquid chromatography-mass spectrometry (LC-MS). However, the use of proteases caused a profound imbalance in the generated peptides. Here, we employed partial acid hydrolysis to achieve more efficient and balanced peptide generation. Various stable isotope-labeled oligopeptides were detected after 0.5 h acid hydrolysis, and marked enhancement of peptide generation compared with the previous enzymatic method was observed, especially for Hyp-Gly (27.8 ± 0.6 ng/μg vs 0.231 ± 0.02 ng/μg). The acid hydrolysate was then heated to generate labeled cyclic dipeptides. Using the novel internal standard mixture in LC-MS, we were able to simultaneously quantitate 23 collagen-derived oligopeptides in human plasma and urine after oral administration of collagen hydrolysate.

Kazunori Mizuno

Nippi Research Institute of Biomatrix

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates