PMC

(A) Ectatic corneas with ultrastructural damages, (B) Crosslinking without soluble collagen happens between proteoglycan and collagen fibrils, and also between collagen molecules, and proteoglycan interfigril; (C) Crosslinking with soluble collagen happens not only between native collagen fibril and proteoglycans, but also between soluble collagen and native collagen, and soluble collagen and proteoglycan.

(A-H) Collagen type I remodeling by microencapsulated-hMSCs after 24 hours of incubation. Green: Bovine DQ FITC collagen type I 10 mg/ml, Orange: Human type I collagen, Red: Rat type I collagen 1 mg/ml and Blue: DAPI. (A) Fluorescent staining of bovine DQ FITC type I collagen, which was mixed with rat type I collagen during fabrication of microspheres and it fluoresces if degraded; (B) Immunofluorescent staining of human type I collagen, which should be synthesized by hMSC; (C) Immunofluorescent staining of rat type I collagen; (D) DAPI, which labelled the nuclei; (E) Merged panels A+B+D (cell nuclei in degrading DQ collagen and newly synthesized human type I collage); (F) Merged panels B+C+D (cell nuclei in starting material rat collagen and newly synthesized human type I collagen); (G) Merged panels A+C+D (starting materials rat type I collagen and DQ collagen are largely co-localizing); (H) Merged panels A+B+C+D (cells synthesizing human type I collagen in starting materials, which are undergoing degradation); (I-N) Collagen type II deposition in MSC-collagen type I microsphere after chondrogenic differentiation for 21 days; (I) Immunofluorescent staining of rat type I collagen, which is the starting material of the microsphere; (J) Immunohistochemistry of human type II collagen (DAB: substrated), which is newly synthesized by MSC during chondrogenic differentiation; (K) Merged panels (I+J); (L) Alexa fluor 488 labelled rat type I collagen; (M) Immunofluorescent staining of human type II collagen; (N) Merged panels (L+M) showing co-localization in some regions.

(A) Western blot analysis of collagen and α-smooth muscle actin (α-SMA) protein expression in normal human lung fibroblasts (NHLFs) transfected with siRNA targeting MTHFD2 or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. (B) Quantification of relative Collagen 1 levels in MTHFD2 knockdown NHLFs at the indicated intervals. (C) Western blot analysis of collagen and α-SMA protein expression in NHLFs transfected with siRNA targeting MTHFD1 or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. (D) Quantification of relative Collagen 1 levels in MTHFD1 knockdown NHLFs at the indicated intervals. (E) Western blot analysis of collagen and α-SMA protein expression in NHLFs transfected with siRNA targeting ALDH1L2 or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. (F) Quantification of relative Collagen 1 levels in ALDH1L2 knockdown NHLFs at the indicated intervals. (G) Western blot analysis of collagen and α-SMA protein expression in NHLFs transfected with siRNA targeting MTHFD1L or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. (H) Quantification of relative Collagen 1 levels in MTHFD1L knockdown NHLFs at the indicated intervals. (I) Western blot analysis of collagen and α-SMA protein expression in NHLFs transfected with siRNA targeting both ALDH1L2 and MTHFD1L or nontargeting siRNA. Cells were treated with TGF-β for the indicated intervals. (J) Quantification of relative Collagen 1 levels in ALDH1L2/MTHFD1L knockdown NHLFs at the indicated intervals. Bar graphs represent mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001.

(a) Schematic of the experimental setup for collagen mineralization. Collagen was cast into polyethyleneimine (PEI)- and glutaraldehyde-treated polydimethylsiloxane (PDMS) microwells. Mineralization of collagen was induced by immersing the microwells into a solution containing calcium, phosphate and polyaspartic acid (PAA) for 3 days. (b) Mineral formation was detected using Alizarin Red S (ARS) and von Kossa (VK) staining. Scale bar = 2 mm. (c) FT-IR spectra of collagen and mineralized collagen formed with PAA (MN-collagen). (d) pXRD patterns of collagen and collagen mineralized with and without PAA. (e) Representative SEM and TEM images visualizing the micro- and nano-structure of collagen and collagen mineralized with and without PAA. Collagen was stained with uranyl acetate to show the periodic banding pattern of collagen fibrils. White arrow: Mineral formation within collagen fibrils. Scale bar = 1 μm for SEM and 200 nm for TEM. (f) Young’s moudulus of collagen and MN-collagen measured by AFM-based nanoindentation (n ≥ 14 locations per condition). * indicates significant difference compared to collagen (p < 0.05).