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. 2013 Mar 15;22(6):889-900.
doi: 10.1089/scd.2012.0495. Epub 2012 Dec 16.

Antioxidation of decellularized stem cell matrix promotes human synovium-derived stem cell-based chondrogenesis

Ming Pei  1 , Ying Zhang, Jingting Li, Dongquan Chen
Affiliations

Antioxidation of decellularized stem cell matrix promotes human synovium-derived stem cell-based chondrogenesis

Ming Pei et al. Stem Cells Dev. .

Abstract

Clinical treatment of cartilage defects is challenging due to concomitant post-traumatic joint inflammation. This study was to demonstrate that the antioxidant ability of human adult synovium-derived stem cells (SDSCs) could be enhanced by ex vivo expansion on a decellularized stem cell matrix (DSCM). Microarray was used to evaluate oxidative, antioxidative, and chondrogenic status in SDSCs after expansion on the DSCM and induction in the chondrogenic medium. Hydrogen peroxide (H2O2) was added to create oxidative stress in either expanded SDSCs or chondrogenically induced premature pellets. The effect of H2O2 on SDSC proliferation was evaluated using flow cytometry. Chondrogenic differentiation of expanded SDSCs was evaluated using histology, immunostaining, biochemical analysis, and real-time polymerase chain reaction. Mitogen-activated protein kinase signaling pathways and p21 were compared in the DSCM and plastic-flask-expanded SDSCs with or without H2O2 treatment. We found that expansion on the DSCM upregulated antioxidative gene levels and chondrogenic potential in human SDSCs (hSDSCs), retarded the decrease in the cell number and the increase in apoptosis, and rendered SDSCs resistant to cell-cycle G1 arrest resulting from H2O2 treatment. Treatment with 0.05 mM H2O2 during cell expansion yielded pellets with increased chondrogenic differentiation; treatment in premature SDSC pellets showed that the DSCM-expanded cells had a robust resistance to H2O2-induced oxidative stress. Extracellular signal-regulated kinases 1 and 2 and p38 were positively involved in antioxidative and chondrogenic potential in SDSCs expanded on the DSCM in which p21 was downregulated. DSCM could be a promising cell expansion system to provide a large number of high-quality hSDSCs for cartilage regeneration in a harsh joint environment.

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Figures

FIG. 1.
FIG. 1.
Effect of decellularized stem cell matrix (DSCM) pretreatment on human synovium-derived stem cell (hSDSC) oxidative stress and chondrogenic potential. In the heatmap (A), the expression intensity was log2-transformed and visualized by color ranging from low expression (blue) to high expression (red). The comparisions of Ecell versus Pcell in the genes (>2-fold change) related to oxidative stress and antioxidation (B) and Ep versus Pp in the genes (>2-fold change) related to antioxidation and cartilage markers (C) were clustered by hierarchical clustering methods. Color images available online at www.liebertpub.com/scd
FIG. 2.
FIG. 2.
Effect of DSCM pretreatment on hSDSC expansion under hydrogen peroxide (H2O2). (A) Cell morphology of hSDSCs expanded on either DSCM or Plastic was shown under phase-contrast microscope after treatment with varying concentrations (0, 0.05, 0.5, and 5 mM) of H2O2 for 4 days. Scale bar is 200 μm. (B) The effect of 0.05 mM H2O2 on the proliferation of hSDSCs expanded on either DSCM or Plastic was evaluated using proliferation index by flow cytometry. (C) The effect of 0.05 mM H2O2 on the proliferation of hSDSCs expanded on either DSCM or Plastic was evaluated using cell count (n=8 flasks each group) by a hemocytometer. (D) The effect of 0.05 mM H2O2 on apoptosis of hSDSCs expanded on either DSCM or Plastic was evaluated using flow cytometry. (E) The effect of 0.05 mM H2O2 on the cell cycle of hSDSCs expanded on either DSCM or Plastic was evaluated using flow cytometry. Color images available online at www.liebertpub.com/scd
FIG. 3.
FIG. 3.
Effect of DSCM pretreatment with 0.05 mM H2O2 on expanded hSDSC chondrogenic potential. (A) hSDSCs expanded on either DSCM or Plastic with H2O2 treatment were chondrogenically induced in a pellet culture system for 21 days. Alcian blue (AB) was used to stain sulfated glycosaminoglycans (GAGs). Immunohistochemistry (IHC) staining was used to detect collagen II (Col II). (B) Biochemical analysis was used for DNA and GAG contents in the 21-day chondrogenically induced pellets. Cell proliferation and viability were evaluated using the DNA ratio (DNA content at day 21 adjusted by that at day 0). Chondrogenic index at day 21 was evaluated using a ratio of GAG to DNA. (C) Real-time polymerase chain reaction (PCR) was used to evaluate chondrogenic marker gene expression (SOX9, ACAN, and COL2A1) in day-21 pellets. Data are shown as average±standard deviation (SD) for n=4. *P<0.05, **P<0.01, and ***P<0.001 compared with the corresponding DSCM group. #P<0.05, ##P<0.01, and ###P<0.001 compared with the corresponding group without H2O2 treatment. Color images available online at www.liebertpub.com/scd
FIG. 4.
FIG. 4.
Effect of DSCM pretreatment on expanded hSDSC chondrogenic differentiation with either 0.05 or 0.1 mM H2O2. (A) hSDSCs expanded on either DSCM or Plastic with H2O2 treatment were chondrogenically induced in a pellet culture system for 21 days and treated with H2O2 for the following 7 days. AB was used to stain sulfated GAGs. IHC staining was used to detect Col II. Scale bar is 1 mm. (B) Biochemical analysis was used for DNA and GAG contents in the 28-day chondrogenically induced pellets. Cell proliferation and viability were evaluated using DNA ratio (DNA content at day 28 adjusted by that at day 0). Chondrogenic index at day 28 was evaluated using ratio of GAG to DNA. (C) Real-time PCR was used to evaluate chondrogenic marker gene expression (SOX9, ACAN, and COL2A1) in day-28 pellets. Data are shown as average±SD for n=4. **P<0.01, and ***P<0.001 compared with the corresponding DSCM group. #P<0.05, and ###P<0.001 compared with the corresponding group without H2O2 treatment. $P<0.05 and $$$P<0.001 compared with the corresponding group treated with 0.05 mM H2O2. Color images available online at www.liebertpub.com/scd
FIG. 5.
FIG. 5.
Effect of H2O2 treatment in an either cell expansion or chondrogenic differentiation phase on expanded SDSC chondrogenic hypertrophy. (A, C) hSDSCs expanded on either DSCM or Plastic with 0.05 mM H2O2 treatment were chondrogenically induced in a pellet culture system for 21 days. (B, D) hSDSCs expanded on either DSCM or Plastic were chondrogenically induced in a pellet culture system for 21 days and treated with H2O2 for the following 7 days. IHC staining was used to detect collagen X (A, B). Scale bar is 1 mm. Real-time PCR was used to evaluate hypertrophy marker gene expression (COL10A1) (C, D). Data are shown as average±SD for n=4. ***P<0.001 compared with the corresponding DSCM group. ###P<0.001 compared with the corresponding group without H2O2 treatment. $$$P<0.001 compared with the corresponding group treated with 0.05 mM H2O2. Color images available online at www.liebertpub.com/scd
FIG. 6.
FIG. 6.
Western blot was used to measure the levels of p38 [(A) p-p38/p38] and Erk1/2 [(B) p-Erk/Erk] (the mitogen-activated protein kinase signaling pathway) and the level of p21 (a senescence-associated marker) [(C) p21/β-actin] in the hSDSCs expanded on either DSCM or Plastic after the treatment with 0.05 mM H2O2. β-actin served as an internal control. ImageJ software was used to quantify immunoblotting bands.
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