Expression of Musashi-1 Increases in Bone Healing

Abstract

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.

Keywords: Musashi-1; Runx2; bone healing; mesenchymal stem cells; periostin.

Figure 1

Evolution of the bone fracture callus at days (A) 4 and (B) 7. Hematoxylin-eosin. Original magnification: 4×.

Figure 2

(A) Histomorphological confirmation after 14 days of an effective endochondral and intramembranous repair around a bone fracture callus involving fibrocartilage (red arrow), cartilage (green arrow), and newly formed bone trabecula (yellow arrow). An adequate number of new vessels can be observed (B) as well as normal distribution of osteoclasts (arrow in (C)), and osteocytes (orange arrow in (D)), and osteoblasts (blue arrow in (D)). Masson’s trichrome staining. Original magnification: 4× (A), 10× (B) and 40× (C,D).

Figure 3

Negative detection of musashi-1 at day 14 in (A) unfractured controls in comparison with (B) bone fracture callus, as also represented in Figure 1. Peroxidase-conjugated micropolymer detection. Original magnification: 10×.

Figure 4

(A) Overview of musashi-1 detection in a bone fracture callus (in brown) and detail observations in (B) fusiform mesenchymal cells (green arrow), and immature chondrocytes (red arrow) in the fibrocartilage, (C) negative detection in mature hypertrophic chondrocytes (orange arrow), and (D) intense detection in the nucleus and cytoplasm of osteoblasts (blue arrow), and osteocytes (black arrow) in the newly formed bone. Peroxidase-conjugated micropolymer detection in samples from the 14-day time point. Original magnification: 10× (A), 20× (B,C) and 40× (D).

Figure 5

Graphical representation of the expression of musashi-1 in bone fracture callus (orange line) and unfractured control areas (blue line) over time in (A) osteoblasts, (B) osteocytes, (C) immature chondrocytes and (D) MSCs (mesenchymal stromal cells).

Figure 6

(A) Overview of RUNX2 detection in a bone fracture callus (in brown) and detail observations in (B) fusiform mesenchymal cells (green arrow), and immature chondrocytes (red arrow) in the fibrocartilage, (C) negative detection in mature hypertrophic chondrocytes (orange arrow), and (D) intense detection in the nucleus and cytoplasm of osteoblasts (blue arrow), and osteocytes (black arrow) in the newly formed bone. Peroxidase-conjugated micropolymer detection in samples from the 14-day time point. Original magnification: 10× (A) and 20× (B–D).

Figure 7

Graphical representation of the expression of RUNX2 in bone fracture callus (orange line) and unfractured control areas (blue line) over time in (A) osteoblasts, (B) osteocytes, (C) immature chondrocytes and (D) MSCs (mesenchymal stromal cells).

Figure 8

Staining of consecutive sections at 4 (A,D), 7 (B,E) and 14 days (C,F) for musashi-1 (A–C) and RUNX2 (D–F). Both markers increased over time. In general, the same cell types expressed both musashi-1 and RUNX2. Peroxidase-conjugated micropolymer detection. TB: trabecular bone; IC: immature chondrocytes; MSCs: mesenchymal stromal cells. Original magnification: 10×.

Figure 9

Weak to negative detection of periostin at day 14 in (A) unfractured controls in comparison with (B) bone fracture callus, where it can be observed mainly in the fibrocartilage (green arrow) and in the intertrabecular interstitium (red arrow). Peroxidase-conjugated micropolymer detection. Original magnification: 10×.

Figure 10

Graphical representation of the relative mRNA expression over time in bone fracture callus (orange line) and unfractured control areas (blue line) of (A) Msi1, (B) Postn and (C) Runx2.

Figure 11

Diagram representing the proposed actions of the three molecules under study on MSC differentiation, collagen fibrillogenesis and, ultimately, bone healing. As shown in this manuscript, there is a positive and statistically significant correlation between the expression of the three markers: periostin, RUNX2, and musashi-1. MSC: mesenchymal stromal cell; p21^WAF−1: cyclin-dependent kinase inhibitor 1; TGFß1: transforming growth factor beta 1.

Figure 12

Representative low magnification image of a bone callus showing the approximate areas of each tissue compartment. (A) Hematoxylin-eosin staining. (B) Masson’s trichrome staining. CB: cortical bone; SM: skeletal muscle; black squares: different tissue compartments of bone callus (from left to right: connective tissue, cartilage, and new bone tissue).