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

Abstract

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).

Cryogel Scaffold-Mediated Delivery of Adipose-Derived Stem Cells Promotes Healing in Murine Model of Atrophic Non-Union

“…In vivo , following implantation at 4 weeks, 50% or less of the animals treated with either scaffold only or the sham control had a complete radiographic bridging at 12 weeks. This blunted healing response was consistent with our previous study that established the Col1-tk atrophic non-union model ( Hixon et al, 2021 ). Comparatively, the radiograph analysis showed that the scaffolds seeded with GFP + cells and the scaffolds seeded with cells producing BMP-2 were the only groups that reached complete bridging in all the animals by the end of 12 weeks.…”

“…We recently demonstrated a model of atrophic non-union in Col1-tk mice dosed with GCV ( Hixon et al, 2021 ). Briefly, a previously established protocol was employed to create a full fracture in the right femur of 12-week-old mice ( Gardner et al, 2011 ).…”

“…All the animals were then monitored by weekly radiography (Faxitron) for an additional 2 weeks to confirm a lack of bone formation and healing response, consistent with a non-union (4 weeks following fracture surgery; Figures 1B ,C). Dosing, withdrawal, and revision surgery timepoints were based on the previous work in this mouse line and cover the established bridging window in the control mice ( Garcia et al, 2013 ; Liu et al, 2017 ; Hixon et al, 2021 ).…”

“…Most available models target fracture healing by disrupting the tissues needed for repair through invasive methods such as osteotomy, periosteal stripping, and/or devascularization ( Mills and Simpson, 2012 ; Gómez-Barrena et al, 2015 ; Panteli et al, 2015 ). While such methods do yield an atrophic non-union, they do not model some clinical atrophic non-unions, which occur without extensive tissue damage and are attributed to “failure of biology.” We recently developed a novel murine model of atrophic non-union in the 3.6Col1A1-tk (Col1-tk) mouse, where dosing with the nucleoside analog ganciclovir (GCV) is used to deplete replicating osteoblast lineage cells ( Hixon et al, 2021 ). Briefly, in proliferating cells that express the thymidine kinase (tk) “suicide gene,” GCV is converted to a toxic nucleotide, which is then incorporated into the DNA.…”

“…Notably, because non-dividing cells are spared, the fracture site is still populated by viable cells including, presumably, skeletal progenitors. Once GCV is withdrawn, there is potential for these resident cells to proliferate and contribute to fracture healing, although our prior work showed that this does not happen spontaneously ( Hixon et al, 2021 ). This biologically relevant model allows for the testing of novel intervention techniques that might reactivate the quiescent native cells to rescue an atrophic non-union.…”