Kartogenin induces cartilage-like tissue formation in tendon-bone junction

Abstract

Tendon-bone junctions (TBJs) are frequently injured, especially in athletic settings. Healing of TBJ injuries is slow and is often repaired with scar tissue formation that compromises normal function. This study explored the feasibility of using kartogenin (KGN), a biocompound, to enhance the healing of injured TBJs. We first determined the effects of KGN on the proliferation and chondrogenic differentiation of rabbit bone marrow stromal cells (BMSCs) and patellar tendon stem/progenitor cells (PTSCs) in vitro. KGN enhanced cell proliferation in both cell types in a concentration-dependent manner and induced chondrogenic differentiation of stem cells, as demonstrated by high expression levels of chondrogenic markers aggrecan, collagen II and Sox-9. Besides, KGN induced the formation of cartilage-like tissues in cell cultures, as observed through the staining of abundant proteoglycans, collagen II and osteocalcin. When injected into intact rat patellar tendons in vivo, KGN induced cartilage-like tissue formation in the injected area. Similarly, when KGN was injected into experimentally injured rat Achilles TBJs, wound healing in the TBJs was enhanced, as evidenced by the formation of extensive cartilage-like tissues. These results suggest that KGN may be used as an effective cell-free clinical therapy to enhance the healing of injured TBJs.

Figure 1

The effect of KGN on the proliferation of rabbit BMSC (a) and PTSCs (b) in vitro. BMSCs and PTSCs were cultured with various concentrations of KGN (1 nmol·L⁻¹–5 µmol·L⁻¹) for 3 days. Cell proliferation in both cultures was assessed by determining cell PDT. Increasing KGN concentrations decreased PDT in both BMSCs and PTSCs, indicating higher cell proliferation rates in response to KGN treatments. The data are expressed as mean±s.d. of three independent experiments. Asterisks represent significant differences (P<0.05) between the treatment groups with various KGN concentrations and the control group, or the cells without KGN treatment.

Figure 2

The effect of KGN on the gene expression of rabbit BMSC (a) and PTSC (b) in vitro. Rabbit BMSCs and PTSCs were grown in medium containing KGN at concentrations ranging from 1 nmol·L⁻¹ to 5 µmol·L⁻¹ for 7 days, followed by RNA extraction and qRT-PCR to determine expression levels of chondrogenesis markers aggrecan, collagen II and Sox-9. In both cell types, KGN increased the expression of all three genes in a dose-dependent manner. Data represent mean±s.d. of three independent experiments. Asterisks indicate significant statistical differences (P<0.05) between the different KGN treatments and their respective controls (0 nmol·L⁻¹ KGN).

Figure 3

The effect of KGN on chondrogenic differentiation of rabbit BMSCs and PTSCs in vitro. BMSCs and PTSCs were cultured in medium containing KGN at concentrations ranging from 1 nmol·L⁻¹ to 5 µmol·L⁻¹ for 2 weeks and stained with Safranin O or Alcian blue. Cells without KGN treatment were used as a control (0 nmol·L⁻¹). Staining for both Safranin O and Alcian blue increased with increasing levels of KGN in both cultures indicating that KGN increases chondrogenic differentiation of BMSCs and PTSCs.

Figure 4

Chondrogenic differentiation of rabbit BMSCs and PTSCs cultured in vitro in medium containing KGN. BMSCs and PTSCs were grown in medium with various concentrations of KGN (1 nmol·L⁻¹–5 µmol·L⁻¹). The cells without KGN treatment were used as a control. After being treated for 2 weeks in culture, the cells were immunostained with anti-collagen II and anti-osteocalcin antibodies to determine chondrogenic differentiation. Nuclei were stained with Hoechst fluorochrome 33342 (blue). Chondrogenic differentiation in both cell types increased after the addition of KGN in a dose-dependent manner. Bars=100 µm.

Figure 5

The effect of KGN injection on intact rat patellar tendons in vivo. Intact patellar tendons were injected with 10 µL saline (a, c) or 10 µL of 100 µmol·L⁻¹ KGN (b, d) four times, on days 1, 2, 7 and 14. Rats were then killed on day 15 to assess the effect of KGN on the patellar tendons. Intact tendons exhibit a white glistening appearance (a, arrow). However, after KGN injection, the original patellar tendon tissue is almost indistinguishable from the newly formed tissues, which appear highly vascularized (b). Staining of the corresponding tissue sections with fast green and Safranin O shows dense collagen in the saline-injected control (c), but extensive accumulation of proteoglycans (arrow) interspersed with collagen is observed in the KGN-injected patellar tendon (d).

Figure 6

The effect of KGN injection on the healing of rat ATBJ. Intact Achilles tendon-bone junctions of rats were wounded (1 mm diameter) and then injected with 10 µL saline (c, e) or 10 µL of 100 µmol·L⁻¹ KGN four times, on days 1, 2, 7 and 14. The rats were killed on day 15 for examination. (a) Intact Achilles tendon–bone junction; (b) wounded ATBJ with a hole indicated by arrow; (c) natural healing of the wounded ATBJ 15 days after wounding; arrow indicates immature, yellowish cartilage-like tissue; (d) completely healed KGN-injected ATBJ 15 days after wounding; arrow points to large, glistening new cartilage-like tissue; (e) saline-injected wound site at the ATBJ stained with fast green and Safranin O shows the lack of proteoglycans in the wound area indicating the absence of fibrocartilage-like tissue formation in the junction; arrow points to the wounded ATBJ area that remained empty even after two weeks of healing. Bone tissues are visible on the left, while tendinous tissues stained with fast green are at the bottom right corner; (f) staining of the KGN-injected ATBJ wound with fast green and Safranin O shows formation of cartilage-like tissue; the wounded ATBJ appears to be filled with abundant proteoglycans stained with Safranin O (triangle), and tendinous tissues are located in the right and appear green with fast green staining. ATBJ, Achilles tendon–bone junction.