Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects

Abstract

This study explored the feasibility of inducing the differentiation of BMSCs into chondrocytes through co-culture with chondrocytes in hydrogel constructs (Pluronic F-127 gel) in vivo for the repair of goat mandibular condylar cartilage defects. Chondrocytes and BMSCs were isolated from goat auricular cartilage and bone marrow, respectively, and were mixed at a ratio of 3:7. BMSCs were labelled with green fluorescence protein (GFP) using a retrovirus vector for tracing. Mixed cells were re-suspended in 30% Pluronic F-127 at a concentration of 5×10⁷ cells/ml to form a gel-cell complex. The gel-cell complex was implanted into the temporomandibular joint condylar articular cartilage defects. The whole temporomandibular joint and adjacent tissues were harvested at 4, 8, and 12 weeks after surgery, and gross observation, histology and collagen II expression were evaluated. In the co-culture group, cartilage-like tissues were formed, and abundant type II collagen could be detected by immunohistochemistry in the condylar cartilage defects. Confocal microscopy revealed that implanted GFP-labelled BMSCs were embedded in cartilage-like tissues. The co-culture system described herein provides a chondrogenic microenvironment to induce the chondrogenic differentiation of BMSCs in vivo without any additional cellular factors.

Keywords: articular; bone marrow stromal cells; condylar; temporomandibular joint.

Figure 1.

Experimental schematic. Chondrocytes and BMSCs were isolated from goat auricular cartilage and bone marrow, respectively, mixed with 30% Pluronic F-127 to form a gel-cell complex and implanted into the TMJ condylar articular cartilage defects. (A and B) The white arrow indicates that the full-thickness condylar cartilage was totally removed and that a large hole was created in the condyle surface. (C) The blue arrow indicates that the gel-cell complex was implanted into the condylar cartilage defects. BMSCs, bone marrow stromal cells; TMJ, temporomandibular joint.

Figure 2.

Characteristics of BMSCs and chondrocytes. BMSCs began to adhere within 24 h after inoculation. (A) BMSCs were spindle-shaped or scalene triangle-shaped (phase contrast, ×100). Newly isolated chondrocytes were rounded, with strong reflection. Cells were adherent to the dish within 3 days. (B) Adhered cells were shaped like transparent polygons with great refractivity (phase contrast, ×100).

Figure 3.

Monitor repaired defects post-surgery by gross view and x-ray. In gel-cell complex implantation group: (A) At week 4, the surfaces were slightly rough and depressed in gross view, and no Pluronic F-127 gel was found on the defect areas (a); (B) at week 8, the surfaces were smooth and a cartilage-like layer can be observed on the surface; (C) at week 12, the surfaces were smooth and a cartilage-like layer can be observed on the surface. (D-F) The X-ray films showed that there was no obvious bone destruction and no osteophyte was formed. In the gel only group: (G) The surfaces were slightly rough and depressed, and no Pluronic F-127 gel was found; (H) the surfaces were rough and depressed; (I) the surfaces were rough and had some fibrous-like tissues on the surface. (J-L) The X-ray films showed that there was no obvious bone destruction and no osteophyte was formed. In the gel only group, the joint space seemed broadened compared with the gel-cell group (indicated by the arrow, phase contrast, ×100).

Figure 4.

Histological evaluation of defects via HE staining and IHC staining with collagen II. For HE staining in the gel-cell complex group: (A) At week 4, the surfaces were slightly irregular and filled with cartilaginous and fibrous tissue; (B) at week 8, the surfaces were much smoother, and most parts of the defects were filled with cartilaginous and fibrous tissue; (C) and at week 12, the surface became very smooth, and the superficial layer was repaired with cartilage and the deeper layer was remodelled with bone or osseous-like tissue. For HE staining in the gel only group: (D) At week 4, the surfaces were irregular and filled with fibrous tissue but not cartilaginous and/or osseous tissue; (E) at week 8, the surfaces were still irregular, and most parts of the defects were filled with fibrous or osseous-like tissue; (F) at week 12, the surfaces were smooth and repaired with mostly osseous-like tissue and some fibrous tissue without any cartilage tissue (phase contrast, ×100). For IHC staining in the gel-cell complex group: (G) At week 4, the cartilage layer was stained with type II collagen at a low intensity; (H) at week 8, the cartilage layer was stained with type II collagen at a much higher intensity; (I) at week 12, the cartilage layer was stained with type II collagen at a high intensity. For IHC staining in the gel only group: (J) At week 4, type II collagen staining was negative, and no Pluronic F-127 gel was found on the surface. The same was observed at (K) 8 weeks and (L) 12 weeks post-surgery. HE, hematoxylin and eosin; IHC, immunohistochemical.

Figure 5.

Evaluation of cartilage repairby confocal microscopy. (A) Quanfication of GFP expression in BMSCs. (a) GFP expression distribution of BMSCs in fluorescent microscopy (×100). (b) GFP-expressed BMSCs selected by flow cytometry. (B) Repaired cartilage comparison between gel-cell complex implantation and control groups were demonstrated by (a and b) confocal microscopy (×200, (c and d) bright field images and (e and f) merged images. GFP-expressed BMSCs were evenly distributed in repaired cartilage area and formed typical lacuna structures at 12 weeks post-surgery. GFP, green fluorescent protein; BMSCs, bone marrow stromal cells.