TY - JOUR
T1 - Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model
AU - Hixon, Katherine R.
AU - McKenzie, Jennifer A.
AU - Sykes, David A.W.
AU - Yoneda, Susumu
AU - Hensley, Austin
AU - Buettmann, Evan G.
AU - Zheng, Hongjun
AU - Skouteris, Dimitrios
AU - McAlinden, Audrey
AU - Miller, Anna N.
AU - Silva, Matthew J.
N1 - Funding Information:
This work was supported by funding from NIAMS (R01 AR050211, P30 AR057235, R21 AR076636‐01, T32 AR060719, and F32 AR076191‐01). We thank the Washington University in St. Louis Musculoskeletal Research Center (MRC) Cores and staff for assistance. Specifically, thanks to Yung Kim for all X‐ray and μCT (Scanco) acquisition assistance. Also thanks to both Crystal Idleburg and Samantha Coleman for histological processing and sectioning of all specimens. Thanks also to Dennis Oakley of the Washington University in St. Louis Center for Cellular Imaging (WUCCI) Core and Heather Zannit for training and frozen section imaging assistance. Paraffin histological images were taken with the Nanozoomer at Alafi Neuroimaging Core (S10 RR027552). Finally, thanks to Nicole Migotsky for torsion testing and LabVIEW software instruction, as well as Hongjun Zheng and Lisa Lawson for limb bud assay assistance. 3.6Col1a1‐tk mice were kindly provided by the laboratories of Drs. Robert Jilka and Charles O'Brien (University of Arkansas for Medical Sciences, Little Rock, AR, USA).
Funding Information:
This work was supported by funding from NIAMS (R01 AR050211, P30 AR057235, R21 AR076636-01, T32 AR060719, and F32 AR076191-01). We thank the Washington University in St. Louis Musculoskeletal Research Center (MRC) Cores and staff for assistance. Specifically, thanks to Yung Kim for all X-ray and ?CT (Scanco) acquisition assistance. Also thanks to both Crystal Idleburg and Samantha Coleman for histological processing and sectioning of all specimens. Thanks also to Dennis Oakley of the Washington University in St. Louis Center for Cellular Imaging (WUCCI) Core and Heather Zannit for training and frozen section imaging assistance. Paraffin histological images were taken with the Nanozoomer at Alafi Neuroimaging Core (S10 RR027552). Finally, thanks to Nicole Migotsky for torsion testing and LabVIEW software instruction, as well as Hongjun Zheng and Lisa Lawson for limb bud assay assistance. 3.6Col1a1-tk mice were kindly provided by the laboratories of Drs. Robert Jilka and Charles O'Brien (University of Arkansas for Medical Sciences, Little Rock, AR, USA). Authors' roles: Study design: KRH, JAM, AM, and MJS. Study conduct: KRH, DAWS, SY, JAM, AH, HZ, and DS. Data collection: KRH, DAWS, SY, JAM, EGB, HZ, and DS. Data analysis: KRH, DAWS, SY, JAM, AH, and EGB. Data interpretation: KRH, JAM, AM, ANM, and MJS. Drafting manuscript: KRH. Revising manuscript: KRH, JAM, and MJS. Approving final version of manuscript: KRH, DAWS, SY, JAM, AH, EGB, HZ, DS, AM, ANM, and MJS. KRH takes responsibility for integrity of data analysis.
Publisher Copyright:
© 2021 American Society for Bone and Mineral Research (ASBMR).
PY - 2021/11
Y1 - 2021/11
N2 - Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a “failure of biology”. We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) “suicide gene” driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro–computed tomography (μCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo μCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion.
AB - Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a “failure of biology”. We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) “suicide gene” driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro–computed tomography (μCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo μCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion.
KW - BIOMECHANICS
KW - GENETIC ANIMAL MODELS
KW - INJURY/FRACTURE HEALING
KW - PRECLINICAL STUDIES
UR - http://www.scopus.com/inward/record.url?scp=85114306756&partnerID=8YFLogxK
U2 - 10.1002/jbmr.4424
DO - 10.1002/jbmr.4424
M3 - Article
C2 - 34405443
AN - SCOPUS:85114306756
VL - 36
SP - 2243
EP - 2257
JO - Journal of Bone and Mineral Research
JF - Journal of Bone and Mineral Research
SN - 0884-0431
IS - 11
ER -