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. 2019 Aug 24;81(8):1211-1218.
doi: 10.1292/jvms.18-0551. Epub 2019 Jun 4.

Comparison of the effect of growth factors on chondrogenesis of canine mesenchymal stem cells

Kentaro Endo  1 , Naoki Fujita  1 , Takayuki Nakagawa  1 , Ryohei Nishimura  1
Affiliations

Comparison of the effect of growth factors on chondrogenesis of canine mesenchymal stem cells

Kentaro Endo et al. J Vet Med Sci. .

Abstract

Mesenchymal stem cells (MSCs) are proposed to be useful in cartilage regenerative medicine, however, canine MSCs have been reported to show poor chondrogenic capacity. Therefore, optimal conditions for chondrogenic differentiation should be determined by mimicking the developmental process. We have previously established novel and superior canine MSCs named bone marrow peri-adipocyte cells (BM-PACs) and the objective of this study was to evaluate the effects of growth factors required for in vivo chondrogenesis using canine BM-PACs. Spheroids of BM-PACs were cultured in chondrogenic medium containing 10 ng/ml transforming growth factor-β1 (TGF-β1) with or without 100 ng/ml bone morphogenetic protein-2 (BMP-2), 100 ng/ml growth differentiation factor-5 (GDF-5) or 100 ng/ml insulin-like growth factor-1 (IGF-1). Chondrogenic differentiation was evaluated by the quantification of glycosaminoglycan and Safranin O staining for proteoglycan production. The expression of cartilage matrix or hypertrophic gene/protein was also evaluated by qPCR and immunohistochemistry. Spheroids in all groups were strongly stained with Safranin O. Although BMP-2 significantly increased glycosaminoglycan production, Safranin O-negative outer layer was formed and the mRNA expression of COL10 relating to cartilage hypertrophy was also significantly upregulated (P<0.05). GDF-5 promoted the production of glycosaminoglycan and type II collagen without increasing COL10 mRNA expression. The supplementation of IGF-1 did not significantly affect cartilaginous and hypertrophic differentiation. Our results indicate that GDF-5 is a useful growth factor for the generation of articular cartilage from canine MSCs.

Keywords: canine; cartilage; chondrogenesis; growth factor; mesenchymal stem cell.

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Figures

Fig. 1.
Fig. 1.
The diameter of spheroids after 7 and 14 days of chondrogenic differentiation under the presence of bone morphogenetic protein-2(BMP-2) (B), growth differentiation factor-5 (GDF-5) (G), or insulin-like growth factor-1 (IGF-1) (I) combined with transforming growth factor-β1 (TGF-β1) (T). Spheroid diameter in the T+B group was significantly larger than those in the other groups. Groups indicated by different letters show significant differences (P<0.05).
Fig. 2.
Fig. 2.
Quantification of DNA and glycosaminoglycan (GAG) content after 7 and 14 days of chondrogenic induction under the presence of bone morphogenetic protein-2 (BMP-2) (B), growth differentiation factor-5 (GDF-5) (G), or insulin-like growth factor-1 (IGF-1) (I) combined with transforming growth factor-β1 (TGF-β1) (T). (A) The DNA content of spheroids in the T+B group was significantly higher than those in the other groups. (B) Total GAG content in spheroids was significantly higher in the T+B group than in the other groups. (C) To evaluate the efficiency of GAG production, the ratio of GAG to DNA was calculated. GAG/DNA was significantly increased in the T+B and T+G groups on day 14. Groups indicated by different letters show significant differences (P<0.05).
Fig. 3.
Fig. 3.
Safranin O staining and immunohistochemistry for type II, I, and X collagen after 14 days of chondrogenic induction under the presence of bone morphogenetic protein-2 (BMP-2) (B), growth differentiation factor-5 (GDF-5) (G), or insulin-like growth factor-1 (IGF-1) (I) combined with transforming growth factor-β1 (TGF-β1) (T). Spheroids in all groups were strongly stained with Safranin O, and stronger staining was observed in the center of the spheroids. In the T+B group, a Safranin O-negative outer layer was formed. All spheroids expressed type II collagen, especially in the center of the spheroids, and the T+G group showed higher expression of type II collagen. The outer fibroblastic layer of the spheroids in the T+B group showed slight expression of type II collagen. Type I collagen was apparently detected on the outer surface of spheroids in all groups. No type X collagen expression was detected in any group. All scale bars indicate 100 µm.
Fig. 4.
Fig. 4.
Gene expression analysis after 14 days of chondrogenic induction under the presence of bone morphogenetic protein-2 (BMP-2) (B), growth differentiation factor-5 (GDF-5) (G), or insulin-like growth factor-1 (IGF-1) (I) combined with transforming growth factor-β1 (TGF-β1) (T). There was no apparent change in the mRNA expression of SOX9, ACAN, COL2, and COL1. The ratio of COL2 to COL1 (COL2/COL1) was higher in the T+G group, but not significantly. The expression of COL10 was significantly increased in the T+B group. The expression of COL10 was upregulated in the T+G group, but not significantly. Groups indicated by different letters show significant differences (P<0.05).
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