Absence of beta3 integrin accelerates early skeletal repair

Abstract

Integrins are heterodimeric transmembrane proteins that mediate cell-matrix interactions and modulate cell behavior. Beta3 subunit is a component of alphaIIbeta3 and alphaVbeta3 integrins. In this study, we first determined that beta3 transcripts are expressed by cells within fracture calluses at 7 and 10 days after injury in a mouse model. We then analyzed fracture healing in mice deficient of beta3 integrin with molecular, histomorphometric, and biomechanical techniques. We found that lack of beta3 integrin results in an extended bleeding time and leads to more bone formation and accelerated cartilage maturation at 7 days after injury. However, beta3 deficiency does not appear to affect later fracture healing. At days 14 and 21, histological appearance or biomechanical properties of fracture calluses are similar between wild type and mutant mice. We also found that altered fracture healing in beta3-null mice is not associated with accelerated angiogenesis, because no significant difference of length density and surface density of blood vessels in fracture limbs was detected at 3 days after injury between wild type and beta3-null mice. In conclusion, our findings demonstrate that beta3 integrin plays an important role during early fracture healing. Further research is required to determine the underlying mechanisms.

Fig. 1

Expression of β3 integrin in fracture calluses. (A) Safranin O/Fast Green (SO/FG) staining of a fracture callus at 7 days after injury. (B) High magnification of the box in (A) shows fibrous callus tissue. (C) Transcripts of β3 integrin (red) were detected in fibrous callus. (D) Blood vessels are detected in the fibrous callus by PECAM immunostaining. (E) Safranin O/Fast Green (SO/FG) staining of a fracture callus at 10 days after injury. (F) High magnification of the box in (E) shows fibrous callus tissue. (G) Transcripts of β3 integrin (red) were detected in (H) vascularized tissues. Scale bar: A, E = 1mm; B–D, F–H = 200 μm.

Fig. 2

Histomorphometric analysis of fracture healing at 7 days after injury. (A) Total volume of callus tissue. (B) Volume of bone. (C) Volume of cartilage. β3 null mice have significantly more bone than wild type mice. Bars indicate the standard deviation.

Fig. 3

Maturation of cartilage at 7 days after fracture. (A–D) wild type mice. (A) Safranin O/Fast Green staining shows cartilage formation (red) in fracture calluses. (B) High magnification of the box in (A) shows a cartilage island. (C) Transcripts of collagen type 2 (Col 2, yellow) are detected in the cartilage island. (D) Part of Col 2 domain is positive for collagen type 10 (Col 10, purple), a marker of hypertrophic chondrocytes. (E–H) β3 null mice. (E) Low and (F) high magnification of fracture callus. (G) Col 2 is expressed by chondrocytes. (H) The majority of Col 2 positive cells are expressing Col 10. Scale bar: A, E = 2mm. B–D, F–H = 100 μm.

Fig. 4

Fracture healing at 14 and 21 days after injury. Safranin O/Fast Green staining did not reveal significant differences in fracture healing between wild type (A, B) and β3-null mice (C, D) at 14 and 21 days after injury. This is further confirmed by histomorphometric analysis (E, F). Scale bar: A–D = 2mm. Bars indicated standard deviation.