Exposure to a youthful circulaton rejuvenates bone repair through modulation of β-catenin

The capacity for tissues to repair and regenerate diminishes with age. We sought to determine the age-dependent contribution of native mesenchymal cells and circulating factors on in vivo bone repair. Here we show that exposure to youthful circulation by heterochronic parabiosis reverses the aged fracture repair phenotype and the diminished osteoblastic differentiation capacity of old animals. This rejuvenation effect is recapitulated by engraftment of young haematopoietic cells into old animals. During rejuvenation, β-catenin signalling, a pathway important in osteoblast differentiation, is modulated in the early repair process and required for rejuvenation of the aged phenotype. Temporal reduction of β-catenin signalling during early fracture repair improves bone healing in old mice. Our data indicate that young haematopoietic cells have the capacity to rejuvenate bone repair and this is mediated at least in part through β-catenin, raising the possibility that agents that modulate β-catenin can improve the pace or quality of fracture repair in the ageing population.

has no effect on rejuvenation but endogenous osteoblasts are required for parabiosis-based rejuvenation. Tibae of 20-month old mice in heterochronic parabiotic pairs were fractured and harvested 14-days post injury. During fracture healing the osteoblasts from either the a, young partner animal or b, the old fractured animal were ablated. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of 25x images represent 400µm and of 200x images represent 100µm. The fracture site is outlined by dashed lines. Bone marrow stromal cells were aspirated from the tibae of unfractured 20-month old mice in isochronic or heterochronic parabiotic pairs, adhered to tissue culture plastic, and differentiated under osteogenic conditions. After 15 days in differentiation media, cultures were washed, fixed, and stained for alkaline phosphatase (ALP) or mineral (Von Kossa). Figure 4 -Young animals retain their robust capacity for fracture repair and osteoblast differentiation. Tibae of 4-month old mice in a, isochronic or b, heterochronic parabiotic pairs were fractured and harvested 14-days post injury. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of 25x images represent 400µm and of 200x images represent 100µm. The fracture site is outlined by dashed lines. Bone marrow stromal cells were aspirated from the tibae of unfractured 4-month old mice in isochronic or heterochronic parabiotic pairs, adhered to tissue culture plastic, and differentiated under osteogenic conditions. After 15 days in differentiation media, cultures were washed, fixed, and stained for alkaline phosphatase (ALP) or mineral (Von Kossa).

Supplementary Figure 5 -Engraftment of young bone marrow rejuvenates fracture repair and osteogenic potential in older animals.
Tibae of 20-month old mice engrafted with a, old and b, young bone marrow were fractured and harvested 14-days post injury. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of 25x images represent 400µm and of 200x images represent 100µm. The fracture site is outlined by dashed lines. c, Amounts of bone, fibrous tissue, and cartilage deposited in the fracture callus was quantified using histomorphometric analysis (5 sections were analysed per fracture callus). Data are expressed as mean +/-95% confidence interval. *Statistically significant, p< 0.05 (Dunnett's test).
Supplementary Figure 6 -Young mice retain robust capacity for fracture repair and osteoblast differentiation. Tibae of 4-month old mice engrafted with a, old or b, young bone marrow were fractured and harvested 21-days post injury. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of 25x images represent 400µm and of 200x images represent 100µm. The fracture site is outlined by dashed lines. Bone marrow stromal cells were aspirated from the tibae of unfractured mice, adhered to tissue culture plastic, and differentiated under osteogenic conditions. After 15 days in differentiation media, cultures were washed, fixed, and stained for alkaline phosphatase (ALP) or mineral (Von Kossa). Figure 7 -Ablation of young donor osteoblasts has no effect but endogenous osteoblasts are required for rejuvenation. Tibae of 20-month old mice engrafted with young bone marrow were fractured and harvested 21-days post injury. During fracture healing osteoblasts from either the a, host or b, the donor animal were ablated. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of 25x images represent 400µm and of 200x images represent 100µm. The fracture site is outlined by dashed lines. Bone marrow stromal cells were aspirated from the tibae of unfractured mice, adhered to tissue culture plastic, and differentiated under osteogenic conditions. After 15 days in differentiation media, cultures were washed, fixed, and stained for alkaline phosphatase (ALP) or mineral (Von Kossa).

Supplementary Figure 8 -Engrafted bone marrow replaces the endogenous CD45+ cell population.
Bone marrow from EYFP+/+ animals was engrafted into wildtype animals and investigated using flow cytometry for EYFP+, CD45+ cells.
Supplementary Figure 9 -The rejuvenation factor contained within conditioned media is heatsensitive. a, Bone marrow stromal cells were aspirated from the tibae of unfractured 4-and 20-month old mice, adhered to tissue culture plastic, and differentiated in osteogenic media conditioned by old cells. After 15 days of differentiation, cultures were washed, fixed, and stained for alkaline phosphatase (ALP) or mineral (Von Kossa). Differentiation potential of cultures was quantified by analysing the number of colony forming units (CFU) for ALP (white bars) and Von Kossa (black bars). b, Cells from 20-month old mice were aspirated and adhered to tissue culture plastic. Differentiation media was conditioned by young cells and either heated or untreated before use. After 15 days of differentiation, cultures were washed, fixed, and stained for alkaline phosphatase (ALP). c, Combined young-and old-conditioned media was incubated on tissue culture plastic and wells were stained for ALP. Data are expressed as mean +/-95% confidence interval. * Statistically significant, p< 0.05 (Dunnett's test).
Supplementary Figure 10 -Modulation of β-catenin is required for rejuvenation of fracture repair. a, Unfractured tibiae from old mice and young mice were investigated for β-catenin levels. b, TCF-Lef reporter mice were fractured and fracture calluses were harvested 7-days post fracture. LacZ/βgalactosidase staining in the fracture callus was quantified. c, β-catenin levels were investigated in osteoblastic cultures of young cells (Y), old cells (O), and old cells rejuvenated with conditioned media (*O). d, 20-month old Cre-only control, β-catenin stabilized, and β-catenin null mice were engrafted with young bone marrow. After two months of engraftment, the tibiae of the mice were fractured and calluses were harvested at 21-days post fracture. Radiographic and histologic (Safranin-O/Fast Green) analysis were used to investigate the progression of tissue repair. Scale bars of images represent 100µm. Results were compared to e, 20-month old Cre-only control, β-catenin stabilized, and β-catenin null mice which did not undergo bone marrow transplantation.
Supplementary Figure 11 -Ad-Dkk-1 protein is expressed during early stages of fracture repair. The tibiae of old mice were treated with Dkk-1-expressing adenovirus and fractured. Fracture calluses were harvested 3-and 14-days post injury. Expression of His-tagged Ad-Dkk-1 was confirmed by investigating protein lysates of fracture calluses using anti-His antibody.