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

Gurpreet S Baht, David Silkstone, Linda Vi, Puviindran Nadesan, Yasha Amani, Heather Whetstone, Qingxia Wei, Benjamin A Alman, Gurpreet S Baht, David Silkstone, Linda Vi, Puviindran Nadesan, Yasha Amani, Heather Whetstone, Qingxia Wei, Benjamin A Alman

Abstract

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.

Figures

Figure 1. Exposure to a youthful circulation…
Figure 1. Exposure to a youthful circulation rejuvenates fracture repair and osteogenic potential in older animals.
(a) Tibae of 20-month-old mice in isochronic (ISO) or heterochronic (HET) parabiotic pairs were fractured and harvested 14 days post injury. Radiographic and histologic (Safranin-O/Fast Green) analyses were used to investigate the progression of tissue repair (n=8 pairs ISO, n=11 pairs HET). Scale bars of × 25 images, 400 μm and of × 200 images, 100 μm. The fracture site is outlined by dashed lines. (b) Mineral apposition during fracture repair was analysed using calcein labelling (top). The distance between the dye fronts was quantified (bottom). (c) Amounts of bone, fibrous tissue and cartilage deposited in the fracture callus was quantified using histomorphometric analysis (five sections were analysed per fracture callus, n=8 fracture calluses-Iso, n=11 fracture calluses-Het). (d) Blood-sharing of parabiosis pairs was confirmed using FLOW cytometry by observing EYFP+ cells in the circulation of WT mice (n=4–7 mice per group). (e) Immunohistochemistry was used to identify EYFP+ cells (blue) and osteocalcin-expressing cells (brown) in the fracture callus. Scale bar, 50 μm. (f) 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 ALP or mineral (Von Kossa; n=5 triplicates per group). Differentiation potential of cultures was quantified by analysing the number of CFU for ALP (white bars) and Von Kossa (black bars; n=5 triplicates per group). Data are expressed as mean±95% confidence interval. *P<0.05, statistically significant (Dunnett's test).
Figure 2. Engraftment of young bone marrow…
Figure 2. Engraftment of young bone marrow rejuvenates fracture repair and osteogenic potential in older animals.
(a) Tibae of 20-month-old mice engrafted with old and young bone marrow were fractured and harvested 21 days post injury. Radiographic and histologic (Safranin-O/Fast Green) analyses were used to investigate the progression of tissue repair (n=10; Old BM, n=9; young BM). Scale bars of × 25 images, 400 μm and of × 200 images, 100 μm. The fracture site is outlined by dashed lines. (b) Mineral apposition during fracture repair was analysed using calcein labelling (top). The distance between the dye fronts was quantified (bottom). (c) Amounts of bone, fibrous tissue and cartilage deposited in the fracture callus was quantified using histomorphometric analysis (five sections were analysed per fracture callus, n=10 fracture calluses—Old BM, n=9 fracture calluses—Young BM). (d) Immunohistochemistry was used to identify EYFP+ cells (blue) and osteocalcin-expressing cells (brown) in the fracture callus. Scale bar, 50 μm. (e) Bone marrow stromal cells were aspirated from the tibae of unfractured 20-month-old mice engrafted with old and young bone marrow. Cells were adhered to tissue culture plastic and differentiated in osteogenic media. After 15 days in differentiation media, cultures were washed, fixed and stained for ALP or mineral (Von Kossa; n=5 triplicates per group). (e) Differentiation potential of cultures was quantified by analysing the number of CFU for ALP (white bars) and Von Kossa (black bars; n=5 triplicates per group). Data are expressed as mean±95% confidence interval. *P<0.05, statistically significant (Dunnett's test).
Figure 3. Media conditioned by young BMSCs…
Figure 3. Media conditioned by young BMSCs rejuvenate aged osteogenic potential.
(a) Bone marrow stromal cells were aspirated from the tibae of unfractured 20-month-old mice, adhered to tissue culture plastic and differentiated in osteogenic media. Cells were differentiated in fresh osteogenic media or in osteogenic media first conditioned by young bone marrow cells. After 15 days of differentiation, cultures were washed, fixed and stained for ALP or mineral (Von Kossa; n=9 triplicates per group). (b) Differentiation potential of cultures was quantified by analysing the number of CFU for ALP (white bars) and Von Kossa (black bars; n=5 triplicates per group). (c) After 10 days of differentiation, transcript levels of osteogenic markers were determined from old cells in fresh (white bars) and conditioned (black bars) osteogenic media (n=4 triplicates per group). Data are expressed as mean±95% confidence interval. *P<0.05, statistically significant (Dunnett's test).
Figure 4. β-catenin levels are elevated in…
Figure 4. β-catenin levels are elevated in older mice but lowered through rejuvenation.
(a) Fracture calluses from tibae of 4- and 20-month-old mice were investigated for β-catenin protein levels using western blot analysis. β-catenin levels were quantified relative to actin (loading control—white bars, young; black bars, old; n=5 fracture calluses per group). (b) Fracture calluses from 20-month-old mice in ISO or HET parabiotic pairs were investigated 7-days post fracture. Samples were investigated for β-catenin and active (unphosphorylated) β-catenin protein levels using western blot analysis (n=5 fracture calluses per group) and Axin2 transcript levels using RT-PCR (n=4 fracture calluses per group). Data are expressed as mean±95% confidence interval. *P<0.05, statistically significant (Dunnett's test).
Figure 5. Modulation of β-catenin rejuvenates fracture…
Figure 5. Modulation of β-catenin rejuvenates fracture repair.
Tibae of 20-month-old mice were treated with adenovirus carrying GFP or Dkk-1, and fracture calluses were harvested 21 days post fracture. (a) Modulation of β-catenin 7 days post fracture was verified using western blot analysis. (b) Radiographic and histologic (Safranin-O/Fast Green) analyses were used to investigate the progression of tissue repair (n=7; GFP, n=11; Dkk-1). Scale bars of × 25 images, 400 μm and of × 200 images, 100 μm. The fracture site is outlined by dashed lines. (c) Amounts of bone and fibrotic tissue deposited in the fracture callus was quantified using histomorphometric analysis (five sections were analysed per fracture callus, n=7 fracture calluses-GFP, n=11 fracture calluses-Dkk-1). Data are expressed as mean±95% confidence interval. *P<0.05, statistically significant (Dunnett's test).

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