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Discoidin Domain Receptor 1 (DDR1) is a receptor tyrosine kinase that binds to and is activated by collagen(s), including collagen type I. Ddr1 deletion in osteoblasts and chondrocytes has previously demonstrated the importance of this receptor in bone development. In this study, we examined the effect of DDR1 ablation on bone architecture and mechanics as a function of aging. Femurs were collected from female global Ddr1 knockout (KO) and wild-type (WT) mice at 2, 6, and 12 mo of age and analyzed by high-resolution micro-computed tomography (μCT), mechanical testing, and histology. Primary monocytes were collected for in vitro osteoclastogenesis assays. Our studies on younger (2 mo) mice revealed no significant differences between the two genotypes and the microarchitectural and mechanical features had a similar trend as those reported earlier for osteoblast or chondrocyte specific Ddr1 knockdown. At an advanced age (12 mo), significant differences were noted across the two genotypes. μCT analysis showed a decrease in medullary cavity area as well as increased trabeculation in cortical and trabecular bone in the Ddr1 KO vs. WT mice. In addition, Ddr1 KO mouse bones exhibited reduced mechanical properties (lower peak load, yield load, and energy to yield) at 12 mo. Histological analysis revealed reduced osteoclast count in Ddr1 KO femurs at 12 mo with no significant difference in osteocyte count between the genotypes. In vitro, osteoclastogenesis was impaired in Ddr1 KO bone marrow derived cells. These results suggest that DDR1 deficiency adversely impacts osteoclast differentiation and bone remodeling in an age-dependent manner. 

Magnetic force microscopy (MFM) is an atomic force microscopy (AFM)-based technique to map magnetic domains in a sample. MFM is widely used to characterize magnetic recording media, magnetic domain walls in materials, nanoparticles and more recently iron deposits in biological samples. However, conventional MFM requires multiple scans of the samples, suffers from various artifacts and is limited in its capability for multimodal imaging or imaging in a fluid environment. We propose a new modality, namely indirect magnetic force microscopy (ID-MFM), a technique that employs an ultrathin barrier between the probe and the sample. Using fluorescently conjugated superparamagnetic nanoparticles, we demonstrate how ID-MFM can be achieved using commercially available silicon nitride windows, MFM probes and AFM equipment. The MFM signals obtained using ID-MFM were comparable to those obtained using conventional MFM. Further, samples prepared for ID-MFM were compatible with multi-modal imaging via fluorescence and transmission electron microscopy. Thus ID-MFM can serve as a high-throughput, multi-modal microscopy technique which can be especially attractive for detecting magnetism in nanoparticles and biological samples. 

Abstract The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson’s trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo , which may have important consequences in the functional role(s) of the underlying ECM.