Naringin and bone marrow mesenchymal stem cells repair articular cartilage defects in rabbit knees through the transforming growth factor-β superfamily signaling pathway

Abstract

The present study aimed to assess the effect of a combination of naringin and rabbit bone marrow mesenchymal stem cells (BMSCs) on the repair of cartilage defects in rabbit knee joints and to assess possible involvement of the transforming growth factor-β (TGF-β) signaling pathway in this process. After establishing an articular cartilage defect model in rabbit knees, 20 New Zealand rabbits were divided into a sham operation group (Sham), a model group (Mod), a naringin treatment group (Nar), a BMSC group (BMSCs) and a naringin + BMSC group (Nar/BMSCs). At 12 weeks after treatment, the cartilage was evaluated using the International Cartilage Repair Society (ICRS)'s macroscopic evaluation of cartilage repair scale, the ICRS's visual histological assessment scale, the Modified O'Driscoll grading system, histological staining (hematoxylin and eosin staining, toluidine blue staining and safranin O staining) and immunohistochemical staining (type-II collagen, TGF-β3 and SOX-9 immunostaining). Using the above grading systems to quantify the extent of repair, histological quantification and macro quantification of joint tissue repair showed that the Nar/BMSCs group displayed repair after treatment in comparison to the untreated Mod group. Among the injury model groups (Mod, Nar, BMSCs and Nar/BMSCs), the Nar/BMSCs group displayed the highest degree of morphological repair. The results of histological and immunohistochemical staining of the repaired region of the joint defect indicated that the BMSCs had a satisfactory effect on the repair of the joint structure but had a poor effect on the repair of cartilage quality. The Nar/BMSCs group displayed satisfactory therapeutic effects on both repair of the joint structure and cartilage quality. The expression level of type-II collagen was high in the Nar/BMSCs group. Additionally, staining of TGF-β3 and SOX-9 in the Nar/BMSCs group was the strongest compared with that of any other group in the present study. Naringin and/BMSCs together demonstrated a more efficient repair effect on articular cartilage defects in rabbit knees than the use of either treatment alone in terms of joint structure and cartilage quality. One potential mechanism of naringin action may be through activation and continuous regulation of the TGF-β superfamily signaling pathway, which can promote BMSCs to differentiate into chondrocytes.

Keywords: bone mesenchymal stem cells; cartilage defect; knee joint; naringin; rabbit; superfamily signaling pathway; transforming growth factor-β.

Figure 1

Flow chart describing the structure of experiments performed to evaluate naringin and bone marrow mesenchymal stem cell induced repair of articular cartilage defects in rabbit knees. BMSCs, bone mesenchymal stem cells; ICRS, International Cartilage Repair Society; TGF-3, transforming growth factor-β3; SOX-9, sry-type high-mobility-group box-9.

Figure 2

Gross appearance and assessment results of articular cartilage defects in the rabbit models at 12 weeks. General appearance of rabbit knee joints at 12 weeks post-operation, including the (A) Sham group, (B) Mod group, (C) Nar group, (D) BMSCs group and (E) Nar/BMSCs group. General appearance of rabbit knee joints before the histological and immunohistochemical assessment, including the (F) Sham group, (G) Mod group, (H) Nar group, (I) BMSCs group and (J) Nar/BMSCs group. BMSC, bone marrow mesenchymal stem cells; Mod, model; Nar, naringin.

Figure 3

Histological findings of the repair tissue at articular cartilage defect sites (n=8 knees/group). Scale bars represent 200 µm. Representative H&E staining at 12 weeks in the (A) Sham group, (B) Mod group, (C) Nar group, (D) BMSCs group and (E) Nar/BMSCs group. Toluidine blue staining at 12 weeks in the (F) Sham group, (G) Mod group, (H) Nar group, (I) BMSCs group and (J) Nar/BMSCs group. Safranin-O staining at 12 weeks in the (K) Sham group, (L) Mod group, (M) Nar group, (N) BMSCs group and (O) Nar/BMSCs group. H&E: Continuous pink on the joint surface illustrates that the cytoplasm of the cartilage is stained pink; Toluidine Blue, continuous dark blue on the joint surface indicates the presence of chondrocytes and cartilage matrix; Safranin O, continuous red on the joint surface indicates the cartilage matrix. BMSC, bone marrow mesenchymal stem cells; H&E, hematoxylin and eosin; Mod, model; Nar, naringin.

Figure 4

Immunohistological findings of the repair tissue at articular cartilage defect sites (n=8 knees/group). Scale bars represent 600 µm. Type II collagen immunostaining at 12 weeks in the (A) Sham group, (B) Mod group, (C) Nar group, (D) BMSCs group and (E) Nar/BMSCs group. TGF-β3 immunostaining at 12 weeks in the (F) Sham group, (G) Mod group, (H) Nar group, (I) BMSCs group and (J) Nar/BMSCs group. SOX-9 immunostaining at 12 weeks in the (K) Sham group, (L) Mod group, (M) Nar group, (N) BMSCs group and (O) Nar/BMSCs group. Type II collagen, TGF-β3 and SOX-9 staining: Brown on the joint surface was positive. BMSC, bone marrow mesenchymal stem cells; Mod, model; Nar, naringin; TGF, transforming growth factor β.

Figure 5

Histological evaluation of the tissue sections. (A) Sections were histologically evaluated based on the ICRS macroscopic evaluation of cartilage repair. (B) Sections were histologically evaluated based on the ICRS Visual Histological Assessment Scale. (C) Sections were histologically evaluated based on modified O’Driscoll grading system. Error bars represent 95% confidence interval. ^*P<0.05, BMSC, bone marrow mesenchymal stem cells; ICRS, International Cartilage Repair Society; Mod, model; Nar, naringin.