Midkine-deficiency delays chondrogenesis during the early phase of fracture healing in mice

Abstract

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.

Figure 1. Mdk-deficient mice aged 9 months displayed increased trabecular number and decreased cortical thickness in the femur.

A) Trabecular bone volume to tissue volume ratio assessed by μCT analysis of volume of interest (VOI) 1 in the intact femur. B) Trabecular number assessed by μCT analysis of the VOI 1 in the intact femur. C) Trabecular thickness. D) Representative μCT images of the intact femurs. E) Cortical thickness assessed by μCT analysis of VOI 2 in the intact femur. *Significantly different from wildtype (p<0.05). (n = 6–7 per group).

Figure 2. Flexural rigidity and moment of inertia was significantly reduced in Mdk-deficient mice after 21 days of the healing period.

Biomechanical and μCT analyses of fractured femur after 21 and 28 days of healing. The volume of interest was determined as the whole periosteal callus between the two inner pin holes. A) Relative flexural rigidity of fractured femur in comparison to intact femur at day 21 and B) at day 28. C) Moment of inertia of the periosteal fracture callus in bending axis x at day 21 and D) at day 28. E) Bone volume to total volume fraction of the periosteal fracture callus at day 21 and F) at day 28. *Significantly different from wildtype (p<0.05). (n = 6–7 per group).

Figure 3. Mdk-deficient mice showed significantly less callus cartilage than wildtype mice 10 days after fracture and significantly more cartilage 21 days after fracture.

Histological sections of fractured femurs were analyzed histomorphometrically using A) Safranin O staining at day 10 or B) Giemsa staining at day 21 and C) at day 28. Scale bar: 500 µm. D) Immunohistochemical Mdk staining of the periosteal callus of wildtype mice at day 4, 10 and 21, showing positively stained periosteal cells at day 4, proliferating and hypertrophic chondrocytes at day 10 and Mdk staining at day 21 in areas of new bone formation. C = cortex; P = periost; PC = proliferating chondrocyte; HC = hyperthropic chondrocyte. Representative images are shown. Scale bar: 500 µm at the upper panel and 50 µm at the lower panel. *Significantly different from wildtype (p<0.05). (n = 6–7 per group).

Figure 4. Immunohistochemical staining showing reduced beta-catenin levels in chondrocytes of Mdk-deficient mice.

Sections of fractured femurs from four mice of each time point and genotype group were stained for each antigen and counterstained using hematoxylin. Representative images are shown; C = cortex; PC = proliferating chondrocyte; Ob = osteoblast; V = vessel; OT = osteocyte; scale bar 50 µm; 200-fold magnification. Beta-catenin staining of the periosteal callus at day 10. PECAM staining of the periosteal fracture callus bridging the osteotomy gap showing the endothelial cells of the newly formed vessels in an area of proliferating chondrocytes at day 10. Enpp1 staining of the osteotomy gap at day 10 showing positively stained proliferating chondrocytes. Dmp1 staining of the periosteal callus at day 10 showing positively stained cortex, osteocytes and areas of new bone formation. Osteoblasts were Dmp1 negative. (n = 4 per group).

Figure 5. Presence of macrophages was delayed in Mdk-deficient mice.

A) Immunohistochemical staining for macrophages at days 4 and 10. Representative images showing recruited macrophages in the marrow cavities proximal to the osteotomy gap. M = macrophage; scale bar 50 µm; 200-fold magnification. The number of macrophages was counted in the marrow cavities close to the osteotomy gap B) at day 4 and C) at day 10. D) TRAP-stained sections from fractured femurs were analyzed for the number of osteoclasts per bone perimeter. E) Toluidine-blue-stained sections were analyzed for the number of osteoblasts per bone perimeter and F) Osteoblast surface per bone surface. (n = 5–6 per group).

Figure 6. Mdk is expressed during ATDC5 cell differentiation and Mdk knockdown significantly delayed early chondrogenic differentiation via suppression of Wnt-target genes.

ATDC5 cells were differentiated and gene expression was evaluated using real-time RT-PCR. B2M was used as the housekeeping gene and gene expression values were normalized to the pre-differentiation values (dotted line). ATDC5 cells were incubated in differentiation medium for 5, 7 and 10 days and A) Mdk and B) collagen2a1 gene expression was evaluated using real time RT-PCR. C) ATDC5 cells were incubated with control siRNA or Mdk siRNA for 24 h and subsequently differentiated for 5 days. Mdk knockdown was verified by analyzing Mdk gene expression. Differentiation was analyzed by evaluation of D) aggrecan or E) collagen2a1 gene expression. Beta-catenin signaling was analyzed by evaluation of F) lef1 and G) axin2 gene expression. H) Western blot analysis of Mdk, collagen type 2 and beta-catenin protein expression at days 0 and 5 of differentiation. GAPDH was used as control. *Significantly different from the control values (p<0.05). (n = 6 per group).