A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing

Kaur, Amandeep; Mohan, Subburaman; Rundle, Charles H.

doi:10.1002/ame2.12114

Cited by 7 publications

(8 citation statements)

References 27 publications

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“…46 SAG is, therefore, expected to promote the differentiation of mesenchymal stem cells toward the chondrocyte lineage and the expansion of cartilage, which is normally insufficient in the segmental defect. 38 We confirmed in vitro that SAG21k 47 was very potent in stimulating CcnD1 expression in chondrocytes (Supporting Information: Figure 1); it increased the expression of markers of chondrocyte development but reduced the expression of mature chondrocyte markers, consistent with the proliferation of immature chondrocytes through SHH signaling. As expected, SAG21k treatment also increased the expression of the SHH targets Gli1 and the receptor Ptch1 (Figure 1).…”

Section: Discussionsupporting

confidence: 64%

“…This method adapts the intramedullary stabilization from the closed femur model to stabilize a larger bone defect than the closed fracture but exhibits severely impaired healing that progresses to nonunion. 38 Bony union in a closed fracture of the murine femur normally occurs between 3 and 4 weeks, with fracture callus remodeling then completing healing. Critical-size endochondral bone defects of more than 2.0 mm result in nonunion healing.…”

Section: Surgical Proceduresmentioning

confidence: 99%

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Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair

Rundle

Gomez

Pourteymoor

et al. 2022

Journal Orthopaedic Research

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The increasing incidence of physiologic/pathologic conditions that impair the otherwise routine healing of endochondral bone fractures and the occurrence of severe bone injuries necessitate novel approaches to enhance clinically challenging bone fracture repair. To promote the healing of nonunion fractures, we tested an approach that used two small molecules to sequentially enhance cartilage development and conversion to the bone in the callus of a murine femoral segmental defect nonunion model of bone injury. Systemic injections of smoothened agonist 21k (SAG21k) were used to stimulate chondrogenesis through the activation of the sonic hedgehog (SHH) pathway early in bone repair, while injections of the prolyl hydroxylase domain (PHD)2 inhibitor, IOX2, were used to stimulate hypoxia signaling-mediated endochondral bone formation. The expression of SHH pathway genes and Phd2 target genes was increased in chondrocyte cell lines in response to SAG21k and IOX2 treatment, respectively. The segmental defect responded to sequential systemic administration of these small molecules with increased chondrocyte expression of PTCH1, GLI1, and SOX9 in response to SAG and increased expression of hypoxia-induced factor-1α and vascular endothelial growth factor-A in the defect tissues in response to IOX2. At 6 weeks postsurgery, the combined SAG-IOX2 therapy produced increased bone formation in the defect with the bony union over the injury. Clinical significance: This therapeutic approach was successful in promoting cartilage and bone formation within a critical-size segmental defect and established the utility of a sequential small molecule therapy for the enhancement of fracture callus development in clinically challenging bone injuries.

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Section: Discussionsupporting

confidence: 64%

Section: Surgical Proceduresmentioning

confidence: 99%

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair

Rundle

Gomez

Pourteymoor

et al. 2022

Journal Orthopaedic Research

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“…( 51 ) It is important to consider potential stress shielding with various models because it can cause asymmetric callus formation or even prevent a periosteal response. ( 52 ) Fourth, it would be of interest to vary the fracture model by both type and location. Although the femur is accepted as appropriate for studying fracture healing, other bones such as the tibia, ulna, rib, radius, and mandible could be useful alternatives.…”

Section: Discussionmentioning

confidence: 99%

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model

Hixon

McKenzie

Sykes

et al. 2021

Journal of Bone and Mineral Research

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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. © 2021 American Society for Bone and Mineral Research (ASBMR).

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“…Other options include intramedullary locking nail or compression screw, external fixator, a pin-clip device, and locking plates [44]. It is important to consider potential stress shielding with various models as it can cause asymmetric callus formation or even prevent a periosteal response [45]. It would also be of interest to vary the fracture model by both type and location.…”

Section: Discussionmentioning

confidence: 99%

“…(52) It is important to consider potential stress shielding with various models as it can cause asymmetric callus formation or even prevent a periosteal response. (53) Fourth, it would be of interest to vary the fracture model by both type and location. While the femur is accepted as appropriate for studying fracture healing, other bones such as the tibia, ulna, rib, radius, and mandible could be useful alternatives.…”

Section: Discussionmentioning

confidence: 99%

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model

Hixon

Sykes

Yoneda

et al. 2020

Preprint

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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. While these approaches can result in nonunion, such invasive methods are 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 old. Post injury, mice were dosed with GCV twice daily for 2 or 4 weeks. Histologically, we confirmed abundant tk+ expression in fracture callus, and diminished periosteal cell proliferation in Col1-tk mice at 3 weeks post fracture. Moreover, Col1-tk mice had less osteoclast activity, mineralized callus, and vasculature at the fracture site compared to WT mice. Additional mice were monitored for 12 weeks with in vivo radiographs and microCT scans, which revealed delayed bone bridging and reduced callus size in Col1-tk mice. Following sacrifice, ex vivo microCT and histology demonstrated failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing demonstrated an inability to recover torsional strength in Col1-tk mice compared to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for either 2 or 4 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.

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A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing

Cited by 7 publications

References 27 publications

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model

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