title: HPLC detection of loss rate and cell migration of HUVECs in a proanthocyanidin cross-linked recombinant human collagen-peptide (RHC)-chitosan scaffold.
abstract: Porous scaffolds with appropriate pore structure, biocompatibility, mechanical property and processability play an important role in tissue engineering. In this paper, we fabricated a recombinant human collagen-peptide (RHC)-chitosan scaffold cross-linked by premixing 30% proanthocyanidin (PA) in one-step freeze-drying. To remove the residual acetic acid, optimized 0.2M phosphate buffer of pH6.24 with 30% ethanol (PBSE) was selected to neutralize the lyophilized scaffold followed by three times deionized water rinse. Ninhydrin assay was used to characterize the components loss during the fabrication process. To detect the exact RHC loss under optimized neutralization condition, high performance liquid chromatography (HPLC) equipped size exclusion chromatography column was used and the total RHC loss rate through PBSE rinse was 19.5±5.08%. Fourier transform infrared spectroscopy (FT-IR) indicated hydrogen bonding among RHC, chitosan and PA, it also presented a probative but not strong hydrophobic interaction between phenyl rings of polyphenols and pyrrolidine rings of proline in RHC. Further, human umbilical vein endothelial cell (HUVEC) viability analyzed by a scanning electron microscope (SEM) and acridine orange/ethidium bromide (AO/EB) fluorescence staining exhibited that this scaffold could not only promote cell proliferation on scaffold surface but also permit cells migration into the scaffold. qRT-PCR exhibited that the optimized scaffold could stimulate angiogenesis associated genes VEGF and CD31 expression. These characterizations indicated that this scaffold can be considered as an ideal candidate for tissue engineering.
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Aipeng
authors.initials: A
authors.lastname: Deng
authors.affiliation: Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Lihu
authors.initials: L
authors.lastname: Gao
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Na
authors.initials: N
authors.lastname: Xu
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Lunxiang
authors.initials: L
authors.lastname: Hu
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
authors.forename: Junhua
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, China. Electronic address: yshulin@njust.edu.cn.
authors.forename: Shulin
authors.initials: S
authors.lastname: Yang
mesh_terms: D002465:Cell Movement; D049109:Cell Proliferation; D048271:Chitosan / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D044945:Proanthocyanidins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D010455:Peptides; D044945:Proanthocyanidins; D011994:Recombinant Proteins; C013221:proanthocyanidin; D003094:Collagen; D048271:Chitosan
keywords: HPLC; HUVEC migration; Hydrogen bonding; Proanthocyanidin; Recombinant human collagen; qRT-PCR