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Fractures in Geriatric Mice Show Decreased Callus Expansion and Bone Volume

  • Basic Research
  • Published:
Clinical Orthopaedics and Related Research®

A CORR Insights to this article was published on 21 August 2014

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.

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Acknowledgments

We thank Michael Karp MS and Brian Horwich MS (micro-CT data acquisition) Department of Bioengineering, University of Pennsylvania; Derek Dopkin BA (colony management) Department of Clinical Studies-New Bolton Center, University of Pennsylvania; Allison Williams BS (quantitative PCR) Department of Clinical Studies-New Bolton Center, University of Pennsylvania; and Michael Dishowitz PhD (experimental design and techniques) Department of Clinical Studies-New Bolton Center, University of Pennsylvania for their technical contributions.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 2 Silverstein, 3400 Spruce Street, Philadelphia, PA, 19104, USA

    Luke A. Lopas MD, Nicole S. Belkin MD, Patricia L. Mutyaba BS, Chancellor F. Gray MD, Kurt D. Hankenson DVM, MS, PhD & Jaimo Ahn MD, PhD

  2. Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, Kennett Square, PA, USA

    Patricia L. Mutyaba BS & Kurt D. Hankenson DVM, MS, PhD

Authors
  1. Luke A. Lopas MD
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  2. Nicole S. Belkin MD
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  3. Patricia L. Mutyaba BS
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  4. Chancellor F. Gray MD
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  5. Kurt D. Hankenson DVM, MS, PhD
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  6. Jaimo Ahn MD, PhD
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Corresponding author

Correspondence to Jaimo Ahn MD, PhD.

Additional information

One or more of the authors certify that they have received, during the study period, funding from NIH R03AG040670 (JA, KDH), American Geriatric Society Jahnigan Award (JA), McCabe Foundation Pilot Award (JA, KDH), and Penn Center for Musculoskeletal Disorders Pilot Award (NIH P30AR050950) (JA, KDH). One of the authors (KDH) certifies that he or she, or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount of less than USD 10,000 from Skelegen (Philadelphia, PA, USA). One of the authors (JA) certifies that he or she, or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount less than USD 10,000 from Synthes Inc (West Chester, PA, USA), an amount of USD 10,000 to USD 100,000 from Merck & Co, Inc (Whitehouse Station, NJ, USA), and an amount less than USD 10,000 from U&I Corp (Uijeongbu-si, Gyeonggi-do, Korea).

All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research ® editors and board members are on file with the publication and can be viewed on request.

Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.

This work was performed at University of Pennsylvania, Philadelphia, PA, USA.

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Lopas, L.A., Belkin, N.S., Mutyaba, P.L. et al. Fractures in Geriatric Mice Show Decreased Callus Expansion and Bone Volume. Clin Orthop Relat Res 472, 3523–3532 (2014). https://doi.org/10.1007/s11999-014-3829-x

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  • DOI: https://doi.org/10.1007/s11999-014-3829-x

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