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. 2018 Mar 15:69:71-82.
doi: 10.1016/j.actbio.2017.12.043. Epub 2018 Jan 6.

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo

Yuanheng Yang  1 , Hang Lin  2 , He Shen  3 , Bing Wang  2 , Guanghua Lei  4 , Rocky S Tuan  5
Affiliations

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo

Yuanheng Yang et al. Acta Biomater. .

Abstract

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo.

Statement of significance: Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.

Keywords: Bone marrow mesenchymal stem cells; Chondrocyte expansion; Chondrogenesis; Extracellular matrix; In vivo cartilage formation; Micromass; Redifferentiation.

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Figures

Figure 1
Figure 1
(A) Schematic representation of preparation of chondrocyte-impregnated hBMSC-ECM. (B) Bright field images of chondrocytes growing on TCP and ECM for 5 days or 2 days. (C) chondrocyte growth curve on TCP or ECM. Chondrocytes were seeded initially at the same density (2,000 cells/cm2, shown by down arrows). After proliferation on TCP for 10 days, chondrocytes reached a cell density similar to those growing on ECM for 6 days (shown by up arrows). Bar = 100 µm in B.
Figure 2
Figure 2
Real time RT-PCR analysis of expression levels of chondrogenic and hypertrophic genes in chondrocytes growing on TCP for 10 days (day 10 TCP), or on ECM for 6 days (day 6 ECM), both of which have undergone similar rounds of proliferation or population doubling. Collagen type II stands for COL2A1, and collagen type I stands for COL1A2. Results are normalized to those in cells prior to seeding, and shown as mean ± SD (*, p<0.05; **, p<0.01).
Figure 3
Figure 3
Representative Western blot analysis of phosphorylated Smad2/3 (p-Smad2/3) and total Smad2/3 (Smad2/3) in chondrocytes after being expanded on TCP for 10 days (day 10 TCP), or on ECM for 6 days (day 6 ECM), with or without TGFβ3 stimulation. Western blots of GAPDH indicate relatively even protein loading among different lanes. The results showed that higher p-Smad2/3 level in day 6 ECM group compared to day 10 TCP group upon chondro-induction.
Figure 4
Figure 4
Assessment of redifferentiation ability of chondrocytes expanded on TCP for 10 days (TCP), or on ECM for 6 days (ECM). Cells were cultured as high-density micromass for 14 days with the treatment of full chondrogenic medium. Chondrogenesis was estimated by histology, real time PCR and biochemical analysis. (A) sGAG of TCP (left) and ECM (right) micromass cultures was detected with Safranin O staining. ECM cultures showed deeper stained sGAG. (B) Real-time PCR analysis of relative mRNA expression levels of chondrogenic (collagen type II and collagen type II/collagen type I) and hypertrophic (collagen type X) genes at different time points. Results are normalized to mRNA level in chondrocytes without culture expansion. ECM micromasses showed higher collagen type II, collagen type II/collagen type I gene expression and considerably lower collagen type X expression. Collagen type II refers to COL2A1, and collagen type I refers to COL1A2. (C) Dimethylmethylene blue dye binding assay and picogreen assay were used to detect sGAG and dsDNA, respectively, and sGAG/dsDNA was calculated. The sGAG/dsDNA ratio of ECM cultures was significantly higher than that of TCP cultures. Data are shown as average ± SD for n = 3. *, p<0.05; ***, p<0.001. Bar=150µm.
Figure 5
Figure 5
Assessment of in vitro chondrogenic capacity of chondrocyte-impregnated hMSC-ECM. Chondrocytes cultured on hMSC-ECM were briefly trypsinized, detached together with the ECM, and cultured for additional 21 days in full chondrogenic medium. The extent of chondrogenesis was estimated histologically. (A, D) Microscopic images of on-ECM chondrocytes with (D) or without (A) trypsin-EDTA treatment. Cells displayed round shapes after the treatment. (B, C, E, F) Safranin O staining of Cell/ECM constructs with (E, F) or without (B, C) trypsin-EDTA treatment after chondro-induction for 21 days. Trypsin+ group showed more compact structure and more uniformly distributed Safranin O staining of sulfated proteoglycan. Bar = 500 µm (B, E) or 150 µm (A, D, C, F).
Figure 6
Figure 6
Assessment of in vitro chondrogenic capacity of chondrocyte-impregnated hMSC-ECM: Gene expression and sGAG analysis. After being expanded on ECM for 6 days, chondrocytes were subjected to 2.5 minutes trypsin-EDTA treatment (Trypsin+), with no treatment as control (Trypsin-). Afterwards, these chondrocytes-impregnated hBMSC-ECM constructs (Cell/ECM constructs) were detached from culture plate and cultured for 21 days of culture in full chondrogenic medium. (A) Real time RT-PCR analysis of mRNA expression levels of chondrogenesis (collagen type II, aggrecan and collagen type I) and hypertrophy (collagen type X, MMP-13) genes at various culture time points, normalized to those of non-culture expanded cells. Collagen type II expression level was higher in the Trypsin+ group, while other genes showed similar expression levels in the Trypsin+ and Trypsin- groups. Collagen type II refers to COL2A1, and collagen type I refers to COL1A2. (B) Dimethylmethylene blue dye binding assay showed significantly higher sGAG content in the Trypsin+ group. ****, p<0.0001.
Figure 7
Figure 7
Schematic of production of cell sheet constructs and Cell/ECM constructs and in vivo cartilage formation testing via subcutaneous implantation in SCID mice. Chondrocytes were thawed and expanded in tissue culture flasks for 6 days and seeded at 10,000 cells/cm2 on TCP and cultured in cell sheet growth medium at day 0. Same number of chondrocytes were expanded for 6 days and seeded at a density of 2,000 cells/cm2 on hBMSC-ECM at day 4. At day 10 of total culture period, folded chondrocytes-impregnated hBMSC-ECM and chondrocytes cell sheets were treated with full chondrogenic medium for an additional 10 days of in vitro culture, and then subcutaneously implanted in SCID mice. At 2 weeks post-implantation, the mice were sacrificed and the implants were excised and processed for different analyses of cartilage formation in vivo.
Figure 8
Figure 8
Characterization of chondrocyte-impregnated hBMSC-ECM constructs used for subcutaneous implantation. Chondrocytes were seeded to form chondrocyte cell sheets (Cell sheet group) or chondrocyte-impregnated hBMSC-ECM (Cell/ECM group) in culture. After 10 days (day 10), RT-PCR and western blotting were performed. Both groups were then folded into 3D constructs. After 10 additional days of in vitro chondro-induction (day 20), RT-PCR assay was performed prior to the constructs being used for subcutaneously implantation. (A) Collagen type II (COL2A1) expression; and (B) aggrecan expression. RT-PCR analysis showed similar levels in the cell sheet and Cell/ECM groups on day 10, but significantly higher collagen type II and aggrecan expression in the Cell/ECM group at day 20. (C) Representative western blot analysis of Smad2/3 activation in cell sheet and Cell/ECM groups upon TGF-β3 treatment. The results showed that the Cell/ECM group displayed higher TGF-β3 dependent p-Smad2/3 level, consistent with higher chondro-induction, compared with the Cell sheet group. Values shown are mean ± SD (n = 3; *, p<0.05; **, p<0.01).
Figure 9
Figure 9
Assessment of in vivo cartilage forming potential of chondrocyte-impregnated hBMSC-ECM constructs. Chondrocytes were cultured on hBMSC-ECM for 6 days to generate chondrocyte-impregnated hBMSC-ECM constructs (Cell/ECM), or for 10 days on TCP to generate chondrocyte cell sheets. After 10 additional days of stimulation with chondrogenic medium, the constructs were subcutaneously implanted, and cartilage formation capacity was examined 2 weeks post-implantation. (A) Collagen type II (COL2A1) and aggrecan gene expression. RT-PCR results, normalized to those prior to culture expansion, showed that Cell/ECM group had significantly higher level of both genes compared with Cell sheet group. (B) Histological analysis of cell sheet (left) and Cell/ECM (right) implants, based on Safranin O staining, showed more abundant and evenly distributed sulfated proteoglycan distribution in the Cell/ECM group compared with the Cell sheet group. Bar = 1 mm. (C) Results from the Dimethylmethylene blue and picogreen based assays showed that the Cell/ECM group had significantly higher sGAG content and sGAG/dsDNA ratio compared with the Cell sheet group. *, p<0.05; **, p<0.01; ****, p<0.0001.
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