Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.

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Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

Approximately 5% to 10% of all bone fractures do not heal completely, contributing to significant patient suffering and medical costs. Even in healthy individuals, fracture healing is associated with significant downtime and loss of productivity. However, no pharmacological treatments are currently available to promote efficient bone healing. A better understanding of the underlying molecular mechanisms is crucial for developing novel therapies to hasten healing. The early reparative callus that forms around the site of bone injury is a fragile tissue consisting of shifting cell populations held together by loose connective tissue. The delicate callus is challenging to section and is vulnerable to disintegration during the harsh steps of immunostaining, namely, decalcification, deparaffinization, and antigen retrieval. Here, we describe an improved methodology for processing early-stage fracture calluses and immunofluorescence labeling of the sections to visualize the temporal (timing) and spatial (location) patterns of cellular and molecular events that regulate bone healing. This method has a short turnaround time from sample collection to microscopy as it does not require lengthy decalcification. It preserves the structural integrity of the fragile callus as the method does not entail deparaffinization or harsh methods of antigen retrieval. Our method can be adapted for high-throughput screening of drugs that promote efficacious bone healing:

According to 2019 statistics, Alzheimer’s affects 5.8 million Americans. It is an undertreated disease with increased prevalence. The microtubule‐associated protein tau (Mapt) gene encodes the Tau protein, which plays a crucial role in the pathology and progression of Alzheimer’s disease in the brain. The Tau protein interacts with microtubules and plays a role in neuronal microtubule stability. Surprisingly, Tau is equally expressed in the heart, skeletal muscle, kidney, adipose, and soft tissue at the protein level, and its function is mostly unknown. Since microtubule stability plays an essential role in cardiac function, we hypothesized that Mapt−/− mice would develop cardiac dysfunction and skeletal muscle function in vivo. In the present study, we investigated both cardiac function and skeletal muscle phenotypes at 12 months of age. Conscious echocardiography (VisualSonics Vevo 2100) and ECG analysis (ECGenie) was performed in male and female Mapt−/−. Mice and compared to age‐ and strain‐matched wildtype controls. Echocardiographic analysis identified an “apparent” increase in Mapt−/− systolic dysfunction compared to wildtype mice (EF%: 83.4±1.9 vs. 79.1±1.5, FS%: 51.5±2.0 vs. 46.9±1.5%, N=6, 7, respectively) at 12 months of age, although both systolic and diastolic LV volumes were significantly decreased in the Mapt−/− mice. Parallel Doppler studies of the mitral valve E/A ratio (1.5±0.4 vs. 2.3±0.5) identified a significant diastolic (relaxation) dysfunction, which may be driving the “apparent” systolic dysfunction and consistent with the clinical parameters of heart failure with preserved ejection fraction (HFpEF). Electrophysiologic analysis of Mapt−/− mice at 12 months of age identified a significantly decreased heart rate (and corresponding increased RR interval), with evidence of an increased heart rate variability (e.g., CV%) indicative of autonomic changes in the heart. No other changes in ECG intervals (i.e., PQ, PR, QRS, QT, ST, QTC ) or ECG amplitudes (SR, R) were identified. These studies demonstrate the Tau protein’s role for the first time in cardiac and skeletal muscle function phenocopying clinically relevant heart failure (HFpEF), characteristic of patients with Alzheimer Disease in recent small studies. Furthermore, these studies indicate that anti‐Tau therapies for Alzheimer Disease currently in development for primary brain disease could potentially have unexpected off‐target effects that should be considered, particularly since heart failure and skeletal muscle weakness in the AD patients is associated common co‐morbidities and mortality. Support or Funding Information Lilly Foundation/Indiana Center for Musculoskeletal Health/ IU School of Medicine, Physician Scientist Initiative, Scientific Research Initiative