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

ABSTRACTNonunion 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 highly 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 PBS. The remainder of mice were dosed with GCV twice daily for 2 or 4 weeks. Histologically, we observed diminished periosteal cell proliferation in Col1-tk mice 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, revealing 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 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.

Transforming Growth Factor Beta (TGF-β) Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Promoting Wnt Signaling Pathway in Atrophic Nonunion

During atrophic nonunion, Wnt signaling pathway is inhibited, resulting in inhibition of BMSC osteogenic differentiation. TGF-β regulates growth and development of the body. However, TGF-β’s effect on osteogenic differentiation of BMSCs in atrophic nonunion has not been reported. The bone tissue and serum of patients with atrophic nonunion and normal healing fractures were collected, and TGF-β mRNA and serum secretion were analyzed by Real time PCR and ELISA. Rat BMSCs were cultured and randomly divided into control group, TGF-β group and TGF-β siRNA group which was transfected with pcDNA-TGF-β plasmid or TGF-β siRNA respectively followed by analysis of cell proliferation by MTT assay, alkaline phosphatase (ALP) activity, Caspase3 activity, expression of RUNX2 and OPN and PPARγ2 mRNA by Real time PCR, and WNT5A and FZD3 expression by Western blot. TGF-β mRNA level and secretion in patients with atrophic nonunion was significantly reduced compared with patients with normal healing fractures (P < 0.05). Transfection of TGF-β siRNA down-regulated TGF-β expression in BMSCs, significantly inhibited cell proliferation, increased Caspase3 activity, decreased ALP activity, RUNX2 and OPN expression, increased PPARγ2 expression and deceased WNT5A and FZD3 expression (P < 0.05). However, transfection of pcDNA-TGF-β plasmid up-regulated TGF-β expression in BMSCs and reversed the above changes (P < 0.05). TGF-β is reduced in atrophic nonunion patients. Targeting TGF-β promotes BMSCs proliferation and osteogenic differentiation by regulating Wnt signaling pathway.

Existence of multilineage mesenchymal progenitor cells in human atrophic nonunion tissue

Background : Though the treatment of atrophic nonunion is often challenging, bone union may occurs in some patients with atrophic nonunion who underwent indirect treatment at the docking site. We aimed at explaining the reasons why does this phenomenon appear? Methods : Five patients diagnosed with atrophic nonunion were enrolled in our study. Nonunion tissues were cut into strips and cultured immediately after being obtained. We got the adherent cells from atrophic nonunion tissues, and applied the flow cytometry to assess the expression of different cell-surface protein. The research in differentiation of the adherent cells was carried out under the induction of various lineage-specific factors, and Western-blot analysis was used to measure the differentiation-related protein expression. Results : We found that the adherent cells from atrophic nonunion tissues have the characteristics similar to bone marrow stromal cells. These traits demonstrated by the flow cytometry consist of positive expression of markers CD29 and CD44, but negative expression of CD34 and CD45. The adherent cells could differentiate into osteogenic, chondrogenic, and adipogenic cells under the different lineage-specific induction factors in vitro. Alkaline Phosphatase (ALP), Col Ⅱ, osteocalcin (OC), SOX9, lipoprotein lipase (LPL) and PPAR-γ2 were high expressed, as measured by Western-blotting. Conclusions : Mesenchymal stem cells obtaining from atrophic nonunion tissues have the potential of transforming into cartilage and bone-forming cells. Furthermore, we get conclusion that the atrophic nonunion tissues play an important role in the healing process.