Fractures in geriatric mice show decreased callus expansion and bone volume

Abstract

Background: Poor fracture healing in geriatric populations is a significant source of morbidity, mortality, and cost to individuals and society; however, a fundamental biologic understanding of age-dependent healing remains elusive. The development of an aged-based fracture model system would allow for a mechanistic understanding that could guide future biologic treatments.

Questions/purposes: Using a small animal model of long-bone fracture healing based on chronologic age, we asked how aging affected (1) the amount, density, and proportion of bone formed during healing; (2) the amount of cartilage produced and the progression to bone during healing; (3) the callus structure and timing of the fracture healing; and (4) the behavior of progenitor cells relative to the observed deficiencies of geriatric fracture healing.

Methods: Transverse, traumatic tibial diaphyseal fractures were created in 5-month-old (n=104; young adult) and 25-month-old (n=107; which we defined as geriatric, and are approximately equivalent to 70-85 year-old humans) C57BL/6 mice. Fracture calluses were harvested at seven times from 0 to 40 days postfracture for micro-CT analysis (total volume, bone volume, bone volume fraction, connectivity density, structure model index, trabecular number, trabecular thickness, trabecular spacing, total mineral content, bone mineral content, tissue mineral density, bone mineral density, degree of anisotropy, and polar moment of inertia), histomorphometry (total callus area, cartilage area, percent of cartilage, hypertrophic cartilage area, percent of hypertrophic cartilage area, bone and osteoid area, percent of bone and osteoid area), and gene expression quantification (fold change).

Results: The geriatric mice produced a less robust healing response characterized by a pronounced decrease in callus amount (mean total volume at 20 days postfracture, 30.08±11.53 mm3 versus 43.19±18.39 mm3; p=0.009), density (mean bone mineral density at 20 days postfracture, 171.14±64.20 mg hydroxyapatite [HA]/cm3 versus 210.79±37.60 mg HA/cm3; p=0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279±46,755 square pixels versus 302,167±137,806 square pixels; p=0.013) and bone content (mean bone volume at 20 days postfracture, 11.68±3.18 mm3 versus 22.34±10.59 mm3; p<0.001) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48±0.10 versus 0.50±0.13; p=0.793), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135+25 versus 90±52; p=0.221) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2±5.3 versus 18.7±5.2; p=0.324) was similar. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25%±6.8% versus 42%±14.5%; p=0.047).

Conclusions: Our findings suggest that the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude.

Clinical relevance: Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.

Fig. 1A–C

Geriatric (25 months) mice show reduced (A) total callus volume and (B) bone volume but similar (C) bone volume fraction (bone volume/total callus volume) relative to young adult (5 months) mice. Micro-CT results of young adult and geriatric C57Bl/6 mice at 10, 15, 20, 25, 30, and 40 days postfracture show that young adult mice form larger calluses with greater total bone volume geriatric mice. Bone volume fraction is increased in young adult mice at 15 and 20 days postfracture. Values are mean ± SD; BV = bone volume; TV = total volume; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2A–H

Three-dimensional reconstructions and midcoronal views of three-dimensional reconstructions of healing fracture callus show delayed, decreased bone formation in geriatric versus young adult mice at (A) 5 months old 10 days postfracture, (B) 5 months old 15 days after fracture, (C) 5 months old 20 days after fracture, (D) 5 months old 40 days after fracture, (E) 25 months old 10 days after fracture, (F) 25 months old 15 days after fracture, (G) 25 months old 20 days after fracture, and (H) 25 months old 40 days after fracture.

Fig. 3A–D

Geriatric mice produce tissue of lower quality during fracture healing. A comparison of micro-CT results between young adult and geriatric C57Bl/6 mice at 15, 20, 25, 30, and 40 days postfracture shows decreased (A) bone and (B) tissue mineral content in geriatric mice relative to young adult mice. (C) Decreased late tissue mineral density occurs in geriatric mice. (D) Increased bone mineral density occurs at early and late times in young adult mice compared with geriatric mice. Values are mean ± SD; HA = hydroxyapatite; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4A–G

Geriatric mice showed delayed endochondral ossification and produced less cartilage and bone than young adult mice. Histomorphometric comparisons of cartilage (safranin O) and bone (Masson’s trichrome) formation at 5, 10, 15, 20, 30, and 40 days postfracture are shown for (A) callus area, (B) cartilage area, (C) hypertrophic cartilage area, (D) percent cartilage, (E) percent hypertrophic cartilage, (F) bone and osteoid area, and (G) percent bone and osteoid. Values are mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5A–H

Comparison views of young adult and geriatric mice at 5, 10, and 20 days postfracture (Stain, safranin O; original magnification, ×10) and 40 days postfracture (Stain, Masson’s trichrome; original magnification; ×10) are shown for (A) 5 months old 5 days, (B) 5 months old 10 days, (C) 5 months old 20 days, (D) 5 months old 40 days, (E) 25 months old 5 days, (F) 25 months old 10 days, (G) 25 months old 20 days, and (H) 25 months old 40 days after fracture.

Fig. 6A–D

Geriatric mice develop an inferiorly organized callus consistent with decreased mechanical properties. Callus architecture was analyzed by micro-CT for young adult and geriatric C57Bl/6 mice at 10, 15, 20, 25, 30, and 40 days postfracture for (A) trabecular number, (B) trabecular thickness, (C) trabecular spacing, and (D) polar moment of inertia. Values are mean ± SD; *p < 0.05; **p < 0.01 ***p < 0.001.

Fig. 7A–C

Geriatric mice showed a reduction in mesenchymal cell proliferation. Immunohistochemistry was performed for proliferating cellular nuclear antigen in (A) undifferentiated mesenchyme (UDM) at 5 and 10 days postfracture, (B) prehypertrophic cartilage at 10 days postfracture, and (C) newly formed bone at 10 and 20 days postfracture for young adult and geriatric mice. Geriatric mice showed a decreased proportion of proliferating cellular nuclear antigen stained undifferentiated mesenchymal cells at 10 days postfracture. Values are mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 8A–B

A comparison of undifferentiated mesenchyme at 10 days postfracture in young adult and geriatric mice is shown. Solid arrows indicate positive (brown) staining cells (Stain, anti-proliferating cellular nuclear antigen; original magnification, ×40). Dashed arrows indicate negative (purple) staining cells (Stain, hematoxylin; original magnification, ×40).