Teriparatide Treatment Improves Bone Defect Healing Via Anabolic Effects on New Bone Formation and Non-Anabolic Effects on Inhibition of Mast Cells in a Murine Cranial Window Model

Abstract

Investigations of teriparatide (rPTH) as a potential treatment for critical defects have demonstrated the predicted anabolic effects on bone formation, and significant non-anabolic effects on healing via undefined mechanisms. Specifically, studies in murine models of structural allograft healing demonstrated that rPTH treatment increased angiogenesis (vessels <30μm), and decreased arteriogenesis (>30 μm) and mast cell numbers, which lead to decreased fibrosis and accelerated healing. To better understand these non-anabolic effects, we interrogated osteogenesis, vasculogenesis and mast cell accumulation in mice randomized to placebo (saline), rPTH (20μg/kg/2days), or the mast cell inhibitor sodium cromolyn (SC) (24μg/kg/2days), via longitudinal micro-CT and multiphoton laser scanning microscopy (MPLSM), in a critical calvaria defect model. Micro-CT demonstrated that SC significantly increased defect window closure and new bone volume versus placebo (p<0.05), although these effects were not as great as rPTH. Interestingly, both rPTH and SC has similar inhibitory effects on arteriogenesis versus placebo (p<0.05) without affecting total vascular volume. MPLSM time course studies in untreated mice revealed that large numbers of mast cells were detected 1 day post-op (43 +/− 17), peaked at 6 days (76 +/− 6), and were still present in the critical defect at the end of the experiment on day 30 (20 +/− 12). In contrast, angiogenesis was not observed until day 4, and functional vessels were first observed on 6 days, demonstrating that mast cell accumulation precedes vasculogenesis. To confirm a direct role of mast cells on osteogenesis and vasculogenesis, we demonstrated that specific diphtheria toxin-α deletion in Mcpt5-Cre-iDTR mice results in similar affects as SC treatment in WT mice. Collectively, these findings demonstrate that mast cells inhibit bone defect healing by stimulating arteriogenesis associated with fibrotic scaring, and that an efficacious non-anabolic effect of rPTH therapy on bone repair is suppression of arteriogenesis and fibrosis secondary to mast cell inhibition.

Mast Cells Are Critical Regulators of Bone Fracture–Induced Inflammation and Osteoclast Formation and Activity

“…In 1967, Lindholm and colleagues described a progressive increase of MCs in the periosteal callus in a rat tibial‐fracture model . This was confirmed by other authors, who found an increased MC number in the early soft callus around blood vessels . Notably, MCs were also observed in the later bony callus near bone resorption sites.…”

Defective bone repair in diclofenac treated C57Bl6 mice with and without lipopolysaccharide induced systemic inflammation

“…repair (Banovac, Renfree, Makowski, Latta, & Altman, 1995;Lindholm et al, 1969) have recently been substantiated (Behrends et al, 2014;Behrends et al, 2017;Ramirez-Garcia-Luna et al, 2017;Zhang et al, 2017). Taken together, these studies suggest that impaired recruitment of mast cells during bone repair leads to disruption of macrophage, vascular endothelial, and bone cell function, culminating in failure to heal.…”

“…Resolution of this acute inflammatory reaction to tissue injury, mediated primarily by neutrophils and M1 macrophages, is arguably the most critical stage of wound healing (Claes, Recknagel, & Ignatius, ). Early reports suggesting that mast cells play a key role in the inflammatory phase of bone repair (Banovac, Renfree, Makowski, Latta, & Altman, ; Lindholm et al, ) have recently been substantiated (Behrends et al, ; Behrends et al, ; Ramirez‐Garcia‐Luna et al, ; Zhang et al, ). Taken together, these studies suggest that impaired recruitment of mast cells during bone repair leads to disruption of macrophage, vascular endothelial, and bone cell function, culminating in failure to heal.…”

Three‐dimensional intravital imaging in bone research

“…The characteristics of the vasculature within the BM is believed to play significant roles in bone biology . Recent IVI studies have visualized the dynamics of a series of characteristics of the vascular network such as the diameter , area , location , and permeability , as shown in Figure A . This is achieved by intravenous perfusion of fluorophore like dextran‐FITC, or more specifically, fluorescently labeled antibodies targeting vascular endothelium .…”

“…However, while angiogenesis (vessels<30 μm) is beneficial to bone healing, arteriogenesis (vessels>30 μm) conduces to fibrotic scaring, indicating a diameter‐dependent effect. Accordingly, Zhang et al underlined the non‐anabolic effect of recombinant parathyroid hormone in bone healing through mast cell inhibition, since mast cells impede bone healing by stimulating arteriogenesis.…”