Table 1.
The antibody dilution ratio for IHC.
Table 2.
The antibody dilution ratio for IF.
Table 3.
The primer sequences of genes tested in real time-PCR analysis.
Table 4.
The primer sequences for SYBR green real time-PCR assays.
Fig 1.
Collagen remodeling in hMSC in rat collagen microspheres.
(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.
Fig 2.
Human MSC-collagen microspheres under cultures.
(A) Temporal change in the size of hMSC-collagen microspheres (n = 10); (B1-5) Live and Dead staining of hMSCs in collagen microspheres at day 21 during chondrogenic differentiation (Green: Live cells labeled by cacein AM; Red: Dead cells labeled ethidium homodimer-1); (C1-5) sox 9 immunofluorescence; (D1-5) Alcian blue staining; (E1-5) Type II collagen immunohistochemistry; (F1-5) Aggrecan immunohistochemistry; (G1-5) Type I collagen immunohistochemistry; (H1-5) Type X collagen immunohistochemistry; 1: Negative control (Normal medium); 2: Positive control (Chondrogenic medium with TGFβ alone); 3: ED (Chondrogenic medium with TGFβ and intracellular protease inhibitor E64D); 4: GM (Chondrogenic medium with TGFβ and extracellular matrix protease inhibitor GM6001); 5: ED+GM (Chondrogenic medium with TGFβ and both intracellular protease inhibitor E64D and extracellular protease inhibitor GM6001).
Fig 3.
Bar charts showing the extracellular matrix composition of hMSC-collagen microspheres after chondrogenesis.
(A) Hydroxyproline (collagen) content (n = 3); (B) Type II collagen content, measured using ELISA (n = 4); (C) Glycosaminoglycan (GAG) content (n = 3); and (D) DNA content (cellularity) (n = 3).
Fig 4.
Bar charts showing the expression level of genes related to chondrogenesis.
(A) SOX9 (gene of sox9); (B) COL2A1 (gene of type II collagen); (C) ACAN (gene of aggrecan); (D) COL1A1 (gene of type I collagen); (E) COLX (gene of type X collagen); (F) MMP13 (gene of matrix metalloprotease 13); (G) MMP2 (matrix metalloprotease 2 or gelatinase A) (n = 3 in triplicates).
Table 5.
Summary on the gene and protein expression of chondrogenic markers in chondrogenically differentiating hMSCs in collagen microspheres under different protease inhibitor treatment.