Coculture of allogenic DBM and BMSCs in the knee joint cavity of rabbits for cartilage tissue engineering

Abstract

The present study aims to assess coculture of allogenic decalcified bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) in the knee joint cavity of rabbits for cartilage tissue engineering. Rabbits were assigned to an in vitro group, an in vivo group, and a blank control group. At the 4th, 8th, and 12th week, samples from all groups were collected for hematoxylin-eosin (HE) staining and streptavidin-peroxidase (SP) method. The morphological analysis software was used to calculate the average absorbance value (A value). SP and flow cytometry demonstrated that BMSCs were induced into chondrocytes. DBM scaffold showed honeycomb-shaped porous and three-dimensional structure, while the surface pores are interlinked with the deep pores. At the 4th week, in the blank control group, DBM scaffold structure was clear, and cells analogous to chondrocytes were scattered in the interior of DBM scaffolds. At the 8^th week, in the in vivo group, there were a large amount of cells, mainly mature chondrocytes, and the DBM scaffolds were partially absorbed. At the 12th week, in the in vitro group, the interior of scaffolds was filled up with chondrocytes with partial fibrosis, but arranged in disorder. In the in vivo group, the chondrocytes completely infiltrated into the interior of scaffolds and were arranged in certain stress direction. The in vivo group showed higher A value than the in vitro and blank control groups at each time point. Allogenic DBM combined BMSCs in the knee joint cavity of rabbits could provide better tissue-engineered cartilage than that cultivated in vitro.

Keywords: Bone marrow derived mesenchymal stem cells; Cartilage tissue engineering; Decalcified bone matrix; Knee joint cavity; Rabbit.

Figure 1. Identification of BMSCs and induction of BMSCs to chondrocytes

(A) The 3rd generation of BMSCs for implantation, when the primary cells were inoculated for 13 days; (B) Morphology of the 3rd generation of BMSCs induced to chondrocytes (×100); (C) Type II collagen immunohistochemistry of the 3rd generation cells using SP method (×100); (D) Type II collagen immunohistochemistry after BMSC induction using SP method (×100); BMSCs, bone marrow-derived mesenchymal stem cells; SP, streptavidin–peroxidase.

Figure 2. Expression of CD105⁺ in BMSCs by flow cytometer

(A) The control group; (B) the 3rd generation of BMSCs; BMSCs, bone marrow-derived mesenchymal stem cells.

Figure 3. Transplantation regions, cell–scaffold compound shape and joint defect repair in each group at different time points after transplantation

(A) Transplantation regions; (B) cell–scaffold compound shape and joint defect repair in each group.

Figure 4. Morphology of cell–scaffold composites in different groups using HE staining at different time points and SEM at the 12th week (B) (×200)

(A) HE staining results; (B) SEM results; the arrow in Figure (A) was the position of scaffold; HE, hematoxylin–eosin; SEM, scanning electron microscope.

Figure 5. Type II collagen immunohistochemistry of all groups using SP method (×200)

SP, streptavidin–peroxidase.