Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease

Abstract

Background: Canine Legg Calvé Perthes disease (LCPD) occurs during the growth period, and the cause of ischemic necrosis of the femoral head during growth remains unclear. If LCPD-affected femoral head-derived mesenchymal stem cells (LCPD-MSCs) can be generated, they can be used as a new tool for the pathophysiological analysis of canine LCPD.

Aim: To generate affected femoral head-derived mesenchymal stem cells (MSCs) from dogs with LCPD and investigate the mRNA expression levels of angiogenesis-related factors and osteogenic differentiation potency of LCPD-MSCs.

Methods: This study was performed using affected femoral heads from dogs diagnosed with LCPD and underwent femoral head and neck ostectomy. The necrotic tissue was harvested from the LCPD-affected femoral head and cultured statically (LCPD group, n = 6). Canine bone marrow-derived MSCs (BM-MSCs) were used as controls (control group, n = 6). First, the morphology of the cultured cells was observed, and the expression of CD29, CD34, CD44, CD45, CD90, and major histocompatibility complex class II was analyzed using flow cytometry. Additionally, the trilineage differentiation potency of the LCPD-affected head-derived adherent cells was examined. Furthermore, the expression levels of HIF1A, VEGFA, VEGFB, and PDGFB mRNAs and the bone differentiation potency of LCPD-affected head-derived adherent cells were investigated.

Results: LCPD-affected femoral head-derived adherent cells showed a fibroblast-like morphology, and the expression of cell surface antigens was similar to that of BM-MSCs. In addition, LCPD-affected femoral head-derived adherent cells showed the same trilineage differentiation potency as BM-MSCs and were consistent with MSC characteristics. Furthermore, the mRNA expression levels of angiogenesis-related factors could be objectively measured in LCPD-MSCs and those MSCs had bone differentiation potency.

Conclusion: In the present study, canine LCPD-MSCs were successfully generated, suggesting their usefulness as a tool for pathological analysis of LCPD in dogs.

Keywords: Angiogenesis related gene; Bone differentiation potency; Dog; Legg Calvé Perthes disease; Mesenchymal stem cell.

Fig. 1.. Harvesting and culturing methods of necrotic tissue from LCPD-affected femoral head. A: Extracted LCPD-affected femoral head was divided through the center of the necrotic area using an electric surgical blade, B: Divided LCPD-affected femoral head, C: Necrotic tissue was harvested with a small bone curette, and D: Aseptic placement of harvested necrotic tissue on a 6-well plate.

Fig. 2.. Morphologies of canine LCPD-affected femoral head-derived adherent cells. A: Day 7, B: Day 10, C: Day 14, Scale bars, 100 μm.

Fig. 3.. Expressions of cell surface antigens as positive marker in canine BM-MSCs and LCPD-affected femoral headderived adherent cells. Flow cytometry analysis revealed that canine BM-MSCs (A–C) and LCPD-affected femoral head-derived adherent cells (D–F) expressed CD29 (A and D), CD44 (B and E), and CD90 (C and F). Blue area: negative isotype control; Red area: samples.

Fig. 4.. Expressions of cell surface antigens as negative marker in canine BM-MSCs and LCPD-affected femoral headderived adherent cells. Flow cytometry analysis revealed that canine BM-MSCs (A–C) and LCPD-affected femoral head-derived adherent cells (D–F) lacked expression of CD34 (A and D), CD45 (B and E), and MHC class II (C and F) expression. Blue area: negative isotype control; red area: samples.

Fig. 5.. Trilineage differentiation potency. Canine LCPD-affected femoral head-derived adherent cells (A-C) and BMMSCs (D-F) differentiated into adipocytes (A and D), osteoblasts (B and E), and chondrocytes (C and F). (A and D): Sudan III staining, (B and E): Alizarin Red staining, and (C and F): Alcian blue staining. Scale bars = 100 μm or 1 mm.

Fig. 6.. mRNA expression levels after bone differentiation of LCPD-MSCs. The expressions of RUNX2, ALPL, and SPP1 mRNAs increased after bone differentiation. A: RUNX2, B: ALPL, and C: SPP1. The data are presented as mean ± standard deviation. *: Significant difference between the two groups (p < 0.05).