assessment-of-bone-fracture-healing-using-micro-computed-tomography

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

musculoskeletal-research-current-methods-and-innovative-approaches

Musculoskeletal Research: Current Methods and Innovative Approaches

ARTICLES DISCUSSED:
Lee J. I., Govindappa, P. K., Wandling, G. D.,&#160;Elfar, J. C. Traumatic peripheral nerve injury in mice. Journal of Visualized ...

The Pennsylvania State University College of Medicine

Pennsylvania State University

MicroRNAs (miRNAs) comprise a class of small non-coding RNAs that regulate the stability and/or translatability of most protein-coding transcripts. Steady-state levels of mature miRNAs can be controlled through mechanisms that influence their biogenesis and/or decay rates. Pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway that promotes the sequence-specific endonucleolytic decay of miRNAs that harbor a CA and/or UA dinucleotide. Here, we describe an in vitro assay for evaluating the susceptibility of AGO2-loaded miRNAs to degradation by different classes of nucleases. This in vitro approach can be used to complement in vivo studies that aim to identify novel miRNA decay factors.

Evaluating the susceptibility of AGO2-loaded microRNAs to degradation by nucleases in vitro.

Impaired fracture healing in patients with obesity-associated type 2 diabetes (T2D) is a significant unmet clinical problem that affects millions of people worldwide. However, the underlying causes are poorly understood. Additionally, limited clinical information is available on how pre-diabetic hyperglycemia in obese individuals impacts bone healing. Here, we use the diet-induced obesity (DIO) mouse (C57BL/6J) model to study the impact of obesity-associated pre-diabetic hyperglycemia on bone healing and fibrillar collagen organization as healing proceeds from one phase to another. We show that DIO mice exhibit defective healing characterized by reduced bone mineral density, bone volume, and bone volume density. Differences in the healing pattern between lean and DIO mice occur early in the healing process as evidenced by faster resorption of the fibrocartilaginous callus in DIO mice. However, the major differences between lean and DIO mice occur during the later phases of endochondral ossification and bone remodeling. Comprehensive analyses of fibrillar collagen microstructure and expression pattern during these phases, using a set of complementary techniques that include histomorphometry, immunofluorescence staining, and second harmonic generation microscopy, demonstrate significant defects in DIO mice. Defects include strikingly sparse and disorganized collagen fibers, as well as pathological accumulation of unfolded collagen triple helices. We also demonstrate that DIO-associated changes in fibrillar collagen structure are attributable, at least in part, to the accumulation of advanced glycation end products, which increase the collagen-fiber crosslink density. These major changes impair fibrillar collagens functions, culminating in defective callus mineralization, remodeling, and strength. Our data extend the understanding of mechanisms by which obesity and its associated hyperglycemia impair fracture healing and underline defective fibrillar collagen microstructure as a novel and important contributor.

Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model.

Osteoarthritis (OA) is the most prevalent degenerative joint disease, resulting in joint pain, impaired movement, and structural changes. As the ability of joint tissue to resist stress is mainly imparted by fibrillar collagens in the extracellular matrix, changes in the composition and structure of collagen fibers contribute to the pathological remodeling observed in OA joints that includes cartilage degeneration, subchondral bone (SCB) sclerosis, and meniscal damage. Using the established OA model of destabilization of the medial meniscus (DMM) in C57BL/6J mice, we performed a comprehensive analysis of the content and structure of collagen fibers in the articular cartilage, subchondral bone, and menisci using complementary techniques, which included second harmonic generation microscopy and immunofluorescence staining. We found that regions exposed to increased mechanical stress in OA mice, typically closest to the site of injury, had increased collagen fiber thickness, dysregulated fiber formation, and tissue specific changes in collagen I and II (Col I and Col II) expression. In cartilage, OA was associated with decreased Col II expression in all regions, and increased Col I expression in the anterior and posterior regions. Col I fiber thickness was increased in all regions with disorganization in the center region. In the superficial SCB, all regions exhibited increased Col I expression and fiber thickness in OA mice; no changes were detected in the deeper regions of the subchondral bone except for increased Col I fiber thickness. In the menisci, OA led to increased Col I and Col II expression in the vascular and avascular regions of the anterior meniscus with increased Col I fiber thickness in these regions. Similar changes were observed only in the vascular region of the posterior meniscus. Our findings provide, for the first time, comprehensive insights into the microarchitectural changes of extracellular matrix in OA and serve as guidelines for studies investigating therapies that target collagenous changes as means to impede the progression of osteoarthritis.

Structural changes in the collagen network of joint tissues in late stages of murine OA.

Maturation of the 3' end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3' untranslated regions (UTRs). The 3'UTR contains motifs that regulate mRNA metabolism; thus, changing the 3'UTR length via APA can significantly affect gene expression. Endochondral ossification is a central process in bone healing, but the impact of APA on gene expression during this process is unknown. Here, we report the widespread occurrence of APA, which impacts multiple pathways that are known to participate in bone healing. Importantly, the progression of endochondral ossification involves global 3'UTR shortening, which is coupled with an increased abundance of shortened transcripts relative to other transcripts; these results highlight the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA exerts this effect by shortening the 3'UTRs of the Col1a1 and Col1a2 mRNAs, thus removing the binding sites of miR-29a-3p, which would otherwise strongly promote the degradation of both transcripts. Taken together, our study is the first to characterize the crucial roles of APA in regulating the 3'UTR landscape and modulating gene expression during fracture healing.

ArticleTotal : 2	Year
Publication title Cited by 8	2022
Publication title	2023

Article	Year
Evaluating the susceptibility of AGO2-loaded microRNAs to degradation by nucleases in vitro. Methods (San Diego, Calif.)\| PubMed ID: 29777751	2019
Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model. Bone\| PubMed ID: 32439570	2020
Structural changes in the collagen network of joint tissues in late stages of murine OA. Scientific reports\| PubMed ID: 35650306	2022
Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing. Bone research\| PubMed ID: 36596777	2023

Reyad A. Elbarbary

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