A novel kartogenin-platelet-rich plasma gel enhances chondrogenesis of bone marrow mesenchymal stem cells in vitro and promotes wounded meniscus healing in vivo

Feng Liu,Hongyao Xu,He Huang

doi:10.1186/s13287-019-1314-x

Feng Liu, Hongyao Xu + Show 1 more

Open Access

https://doi.org/10.1186/s13287-019-1314-x

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Abstract

BackgroundThe meniscus tear is one of the most common knee injuries particularly seen in athletes and aging populations. Subchondral bone sclerosis, irreparable joint damage, and the early onset of osteoarthritis make the injured meniscus heal difficultly.MethodsThe study was performed by in vitro and in vivo experiments. The in vitro experiments were carried out using the bone marrow stem cells (BMSCs) isolated from the rabbits, and the stemness of the BMSCs was tested by immunostaining. The BMSCs positively expressed stem cell markers were cultured with various concentrations of kartogenin (KGN) for 2 weeks. The chondrogenesis of BMSCs induced by KGN was examined by histochemical staining and quantitative RT-PCR. The in vivo experiments were completed by a rabbit model. Three holes were created in each meniscus by a biopsy punch. The rabbits were treated with four different conditions in each group. Group 1 was treated with 20 μl of saline (saline); group 2 was treated with 5 μl of 100 μM KGN and 15 μl saline (KGN); group 3 was treated with 5 μl of 100 μM KGN, 5 μl of 10,000 U/ ml thrombin, and 10 μl of PRP (KGN+PRP); group 4 was treated with 10,000 BMSCs in 10 μl of PRP, 5 μl of saline solution, and 5 μl of 10,000 U/ml thrombin (PRP+BMSC); group 5 was treated with 10,000 BMSCs in 10 μl of PRP, 5 μl of 100 μM KGN, and 5 μl of 10,000 U/ml thrombin (KGN+PRP+BMSC). The menisci were collected at day 90 post-surgery for gross inspection and histochemical analysis.ResultsThe histochemical staining showed that KGN induced chondrogenesis of BMSCs in a concentration-dependent manner. The RT-PCR results indicated that chondrocyte-related genes were also increased in the BMSCs cultured with KGN in a dose-dependent manner. The in vivo results showed that large unhealed wound areas were still found in the wounds treated with saline and KGN groups. The wounds treated with BMSCs-containing PRP gel healed much faster than the wounds treated without BMSCs. Furthermore, the wounds treated with BMSCs-containing KGN-PRP gel have healed completely and formed more cartilage-like tissues than the wounds treated with BMSCs-containing PRP gel.ConclusionsBMSCs could be differentiated into chondrocytes when they were cultured with KGN-PRP gel in vitro and formed more cartilage-like tissues in the wounded rabbit meniscus when the wounds were treated with BMSCs-containing KGN-PRP gel. The results indicated that the BMSCs-containing KGN-PRP gel is a good substitute for injured meniscus repair and regeneration.

Highlights

The meniscus tear is one of the most common knee injuries seen in athletes and aging populations
When P values are less than 0.05, the two groups compared are considered to be significantly different. Both in vitro and in vivo experiments first required the isolation of Bone marrow-derived stem cell (BMSC) from rabbit femur bones, and the stemness of these BMSCs was confirmed by colony formation, multi-differentiation potential, and stem cell marker expression
Immunostaining indicated the cells isolated from the rabbit femur bones were stem cells as evidenced by more than 90% of BMSCs expressed nucleostemin (Fig. 3a–c, m), more than 63.43% of BMSCs expressed stro-1 (Fig. 3d–f, m), more than 78% of BMSCs were positively stained by CD44 (Fig. 3g–i, m), and more than 96% of BMSCs were positively stained with CD90 (Fig. 3j–l, m)

Summary

Introduction

The meniscus tear is one of the most common knee injuries seen in athletes and aging populations. The meniscus was dissected into inner and outer zones. The structure and composition of the two zones are different. The inner zone of the meniscus is an avascular/aneural region (whitewhite zone) with an articular cartilage-like structure, and the cells produce predominantly type II collagen and proteoglycans. The outer zone of the meniscus is a vascular/ neural region (red-red zone) with a fibrocartilage-like structure and composed of cells with a higher proportion of type I collagen. These two areas are separated by the red-white region, which presents attributes from both redred and white-white regions. The anatomical difference in the vascular supply limits the ability of meniscus to heal. It has been reported that the healing capacity of each area is directly related to blood circulation, leaving the white region susceptible to permanent post-traumatic and degenerative lesions [4]

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