Human extracellular matrix (ECM)-like collagen and its bioactivity

Abstract

Collagen, the most abundant structural protein in the human extracellular matrix (ECM), provides essential support for tissues and guides tissue development. Despite its widespread use in tissue engineering, there remains uncertainty regarding the optimal selection of collagen sources. Animal-derived sources pose challenges such as immunogenicity, while the recombinant system is hindered by diminished bioactivity. In this study, we hypothesized that human ECM-like collagen (hCol) could offer an alternative for tissue engineering. In this study, a facile platform was provided for generating hCol derived from mesenchymal stem cells with a hierarchical structure and biochemical properties resembling native collagen. Our results further demonstrated that hCol could facilitate basal biological behaviors of human adipose-derived stem cells, including viability, proliferation, migration and adipocyte-like phenotype. Additionally, it could promote cutaneous wound closure. Due to its high similarity to native collagen and good bioactivity, hCol holds promise as a prospective candidate for in vitro and in vivo applications in tissue engineering.

Keywords: cell-derived matrix; extracellular matrix; hierarchical structure; native-like collagen.

Graphical abstract

Figure 1.

hTERT-hMSC growth and morphology of PES scaffold and ECM. (A) Representative confocal 3D scanning images of the apical surface (top view), the basal surface (bottom view) and side view of PES scaffold subjected to staining for hTERT (green) after culturing for 2, 6 and 10 days, scale bar= 500 μm. Statistical difference: *P < 0.05 and **P < 0.01 compared with the day 2 (control) when cells start to express hTERT. (B) The relative TERT fluorescent intensity change in PES scaffolds with different culturing days. Data shown are the means $\pm \mathrm{ }$SD for three independent experiments. SEM images of (C) the surface and the cross-section of PES scaffold (day 10), (D) the scaffold surface after decellularization (day 14), (E) ECM after removing PES materials and (F) ECM at higher magnification. The scale bars: 300 μm (C and D), 200 μm (E) and 100 μm (F).

Figure 2.

Biochemical characterization of hCol as compared to native bCol I. (A) The photo of purified massive hCol. (B) Mass spectrum of hCol as a function of molecular weight (MW). (C) Most abundance of collagen chain in hCol as detected by mass proteomics. (D) Protein gel electrophoresis of the collected hCol from the PES scaffolds. Lane 1 is molecular weight ladder and lanes 2 and 3 are from the samples of bCol and hCol. (E) The percentage of O-glycosites and N-glycosites in bCol I and hCol, respectively. (F) The relative abundance of N-glycan composition for N1267 and N1365 between bCol I and hCol (upper pannel: bCol I; lower pannel: hCol).

Figure 3.

The molecular structure analysis of hCol and bCol I. (A) FTIR spectra and (B) Raman spectra of bCol I and hCol. The decomposition of amide I Raman band for (C) bCol I and (D) hCol. (E) The CD spectra of bCol I and hCol at room temperature 22˚C. (F). mean residue molar ellipticity at 222 nm [Θ], as a function of temperature for bCol I (black dot) and hCol (red dot) which is fitted with the sigmoid equation. The first derivatives from sigmoid fits were shown dashed lines.

Figure 4.

The morphology analysis of fibril structure in hCol. TEM images of (A) a cluster of collagen fibrils, (B) individual fibril and (C) D-periodicity within fibrils. (D) The HAADF-STEM image and EDS mapping of the collagen fibril for (E) carbon element and (F) nitrogen element.

Figure 5.

hCol Promotes cell proliferation, cell migration and adiopenesis of hASC. (A) Representative images show cell confluency at different times (blank control: complete growth medium alone; AIM: adipogenic induction medium alone; AIM+ hCol: seeded on hCol-coated surface in AIM medium), scale bar =200 μm. (B) CCK-8 assay: OD450 absorbance in hASCs cells (mean ± SD, n = 4). (C) Cell growth curve determined by cell count after 0, 24, 48 and 72 h of hASC cells exposure to hCol media (mean ± SD, n = 3). (D) representative images of cell migration in different collagen (COL) groups and different treatment groups, scale bar = 50 μm. (E) The cell migration rate of different COL groups (means $\pm \mathrm{ }$SD, n = 3). (F) The migration rate of cells exposure to hCol with different treatments (means $\pm \mathrm{ }$SD, n = 2). (G) The representative images of Oil Red O staining of hASC (4×: scale bar = 200 μm; 20×: scale bar = 50 μm). (H) The quantified result for Oil Red O-positive hASCs cultured in control growth media, AIM and AIM + hCol. The relative expression of (I) LPL and (J) PPARγ2 gene normalized to GAPDH mRNA expression. (mean ± SD, n = 3). Statistical difference: *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared with blank control at each time point.

Figure 6.

The effect of hCol on wound healing in mouse tail skin. (A) Representative photographs of mouse tail skin days 0, 7, 14 and 21 after wounding (control: commercial collagen). (B) The quantified wound closure area normalized to day 0 post-operation. (C) HE staining of cross-section of mouse tail 14 days and 21 days after operation. All bars shown in (B) represents mean ± SD (n = 3, hCol; n = 3, control). *P < 0.05; **P < 0.01 versus control at each time point.