Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification

Abstract

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 Ca²⁺ /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.

Keywords: ANABOLICS; ANIMAL MODELS; INJURY/FRACTURE HEALING; ORTHOPAEDICS; PRECLINICAL STUDIES; THERAPEUTICS.

Figure 1.. CaMKK2 inhibition enhances bone volume and mature bone content of the fracture callus.

(A) Representative weekly radiographic images of mice treated with vehicle (VEH) or STO-609 (n=40/group) taken to monitor the progression of fracture healing. White arrows indicate the initial site of fracture. (Bi) Representative longitudinal micro-CT images of the fracture callus at 14 and 28 days post-fracture (n=6/group). (Bii) Three-dimensional models of the callus derived from micro-CT scans demonstrate the differential thresholding of immature (new callus) bone in gray and mature (fully mineralized) bone in red. (Biii) Cross sections of the previously mentioned 3D models. (C) Mean percent bone volume (BV/TV) of the fracture callus at 14 and 28 days post-fracture, excluding the original cortical bone and marrow cavity. (D) Mean callus maturity calculated as a proportion of immature and mature bone present in the healing fracture callus at 14 and 28 days post-fracture. Error bars represent standard deviation; p-values: * p < 0.05, **** p < 0.0001.

Figure 2.. Treatment with STO-609 accelerates chondrocyte hypertrophy in the fracture callus.

(A-C) Representative images calluses treated with VEH (i, iii; n=3/group) or STO-609 (ii, iv; n=3/group) at days 7 and 14 post-fracture; stained as follows: (A) Cartilage was stained histochemically with safranin O fast green (SOFG; 40X magnification); cartilage - orange; other tissue including bone - green. (B-C) Immunofluorescence (IF) staining (100X magnification) for expression of COL10 (B) and COL2 (C), both in green. Sections were counterstained with DAPI to visualize nuclei as blue. (D-E) Quantitation of mean IF intensity for COL10 (D) and COL2 (E) expression, measured using ImageJ analysis (n=3/group). (F-G) Normalized levels of mRNA for Col10 (F) and Col2a1 (G) relative to Gapdh (n=3/group). Error bars represent standard deviation; p-value: * p < 0.05.

Figure 3.. Early upregulation of OSX and VEGF in the callus following CaMKK2 inhibition.

(A-D) Representative images (200X magnification) of 7-day old calluses treated with VEH (i; n=3) or STO-609 (ii; n=3); stained as follows: (A) VEGF (red): (B) OSX (green) and (C) overlay of VEGF and OSX positive cells (yellow). Sections were counterstained with DAPI to visualize nuclei as blue. (E) Quantitation of mean IF intensity for VEGF and OSX measured using ImageJ analysis (n=3/group). (F) Normalized levels of Osx and Vegf mRNA relative to Gapdh (n=3/group). Error bars represent standard deviation; p-value: * p < 0.05.

Figure 4.. Inhibition of CaMKK2 leads to increased Indian hedgehog signaling.

(A) Representative images (100X magnification) of calluses treated with VEH (i; n=3) or STO-609 (ii; n=3) at day 7 post-fracture; stained for IHH expression in green. Sections were counterstained with DAPI to visualize nuclei as blue. (Aiii) Quantitation of mean IF intensity for IHH measured using ImageJ analysis (n=3/group). (B-D) Normalized levels of Ihh (B), Gli1 (C) and Ptch1 (D) mRNA relative to Gapdh (n=3/group) on days 3 and 7 post-fracture. Error bars represent standard deviation; p-value: ** p < 0.01, **** p < 0.0001.

Figure 5.. STO-609 treatment enhances bone matrix formation at the fracture callus.

(A) Representative images of 7 and 14 day old calluses treated with VEH or STO-609 (n=3/group); stained for expression of COL1A1 (40X and 100X magnification; green). Sections were counterstained with DAPI to visualize nuclei as blue. (B) Quantitation of mean IF intensity for COL1A1 measured using ImageJ analysis (n=3/group). (C) Normalized levels of Col1a1 mRNA relative to Gapdh (n=3/group). Error bars represent standard deviation; p-value: * p < 0.05.

Figure 6.. STO-609 increases bone mineralization at the callus periphery.

(A-B) Representative images of 14 day old calluses stained with Von Kossa and McNeil’s tetrachrome (A; VKM; 50X, 100X, 200X magnification) and picrosirius red (B; PSR; 400X); mineralized bone, black; osteoid, light blue; organization of collagen fibers, red. (n=6/group). (C) Percentage of the peripheral callus bone surface covered with osteoid as measured using histomorphometry (n=6/group). Error bars represent standard deviation; p-value: * p < 0.05.

Figure 7.. CaMKK2 inhibition imparts greater torsional strength on the fracture callus and contralateral mid-shaft.

Torsional strength of fractured and contralateral femurs treated with either VEH or STO-609 (n=10/group) 28 days after fracture was measured as follows: (A) maximum torque to failure in newton-meters of the fracture callus or contralateral mid-shaft; (B) torsional stiffness calculated from the torque-rotation angle curves. Error bars represent standard deviation; p-value: * p < 0.05, ** p < 0.01.

Figure 8.. A model for accelerated bone fracture healing following CaMKK2 inhibition.

This diagram summarizes the events of fracture healing observed in mice treated with vehicle (left) or STO-609 (right). Pharmacological inhibition of CaMKK2 with STO-609 was associated with increased numbers of proliferating (Col2) chondrocytes by day 7 post-fracture. Concurrent expression of high levels of IHH in these calluses correlated with the presence of increased numbers of hypertrophic (Col10) chondrocytes, MSCs (Prx1) and Osx-positive osteoprogenitors along with increased VEGF expression in the central callus. Subsequently, CaMKK2-inhibited animals possessed increased levels of COL1 expression, more organized bone matrix within the callus as well as a greater proportion of mineralized bone along the callus periphery by day 14 post-fracture. By day 28, treatment with STO-609 resulted in enhanced callus maturity and strength compared to control mice, indicating rapid and efficient healing of the bone fracture following CaMKK2 inhibition.