Persistent homology analysis distinguishes pathological bone microstructure in non-linear microscopy images.

We present a topological method for the detection and quantification of bone microstructure from non-linear microscopy images. Specifically, we analyse second harmonic generation (SHG) and two photon excited autofluorescence (TPaF) images of bone tissue which capture the distribution of matrix (fibrillar collagen) structure and autofluorescent molecules, respectively. Using persistent homology statistics with a signed Euclidean distance transform filtration on binary patches of images, we are able to quantify the number, size, distribution, and crowding of holes within and across samples imaged at the microscale. We apply our methodology to a previously characterized murine model of skeletal pathology whereby vascular endothelial growth factor expression was deleted in osteocalcin-expressing cells (OcnVEGFKO) presenting increased cortical porosity, compared to wild type (WT) littermate controls. We show significant differences in topological statistics between the OcnVEGFKO and WT groups and, when classifying the males, or females respectively, into OcnVEGFKO or WT groups, we obtain high prediction accuracies of 98.7% (74.2%) and 77.8% (65.8%) respectively for SHG (TPaF) images. The persistence statistics that we use are fully interpretable, can highlight regions of abnormality within an image and identify features at different spatial scales.

The effects of physiological and injurious hydrostatic pressure on murine ex vivo articular and growth plate cartilage explants: an RNAseq study.

Introduction: Chondrocytes are continuously exposed to loads placed upon them. Physiological loads are pivotal to the maintenance of articular cartilage health, while abnormal loads contribute to pathological joint degradation. Similarly, the growth plate cartilage is subject to various loads during growth and development. Due to the high-water content of cartilage, hydrostatic pressure is considered one of the main biomechanical influencers on chondrocytes and has been shown to play an important role in the mechano-regulation of cartilage. Methods: Herein, we conducted RNAseq analysis of ex vivo hip cap (articular), and metatarsal (growth plate) cartilage cultures subjected to physiological (5 MPa) and injurious (50 MPa) hydrostatic pressure, using the Illumina platform (n = 4 replicates). Results: Several hundreds of genes were shown to be differentially modulated by hydrostatic pressure, with the majority of these changes evidenced in hip cap cartilage cultures (375 significantly upregulated and 322 downregulated in 5 MPa versus control; 1022 upregulated and 724 downregulated in 50 MPa versus control). Conversely, fewer genes were differentially affected by hydrostatic pressure in the metatarsal cultures (5 significantly upregulated and 23 downregulated in 5 MPa versus control; 7 significantly upregulated and 19 downregulated in 50 MPa versus control). Using Gene Ontology annotations for Biological Processes, in the hip cap data we identified a number of pathways that were modulated by both physiological and injurious hydrostatic pressure. Pathways upregulated in response to 50 MPa versus control, included those involved in the generation of precursor metabolites and cellular respiration. Biological processes that were downregulated in this tissue included ossification, connective tissue development, and chondrocyte differentiation. Discussion: Collectively our data highlights the divergent chondrocyte phenotypes in articular and growth plate cartilage. Further, we show that the magnitude of hydrostatic pressure application has distinct effects on gene expression and biological processes in hip cap cartilage explants. Finally, we identified differential expression of a number of genes that have previously been identified as osteoarthritis risk genes, including Ctsk, and Chadl. Together these data may provide potential genetic targets for future investigations in osteoarthritis research and novel therapeutics.

Sexing Bones: Improving Transparency of Sex Reporting to Address Bias Within Preclinical Studies.

Despite knowledge that sexually dimorphic mechanisms regulate bone homeostasis, sex often remains unreported and unconsidered in preclinical experimental design. Failure to report sex could lead to inappropriate generalizations of research findings and less effective translation into clinical practice. Preclinical sex bias (preferential selection of one sex) is present across other fields, including neuroscience and immunology, but remains uninvestigated in skeletal research. For context, we first summarized key literature describing sexually dimorphic bone phenotypes in mice. We then investigated sex reporting practices in skeletal research, specifically how customary it is for murine sex to be included in journal article titles or abstracts and then determined whether any bias in sex reporting exists. Because sex hormones are important regulators of bone health (gonadectomy procedures, ie, ovariectomy [OVX] and orchidectomy [ORX], are common yet typically not reported with sex), we incorporated reporting of OVX and ORX terms, representing female and male mice, respectively, into our investigations around sex bias. Between 1999 and 2020, inclusion of sex in titles or abstracts was low in murine skeletal studies (2.6%-4.06%). Reporting of OVX and ORX terms was low (1.44%-2.64%) and reporting of OVX and ORX with sex uncommon (0.4%-0.3%). When studies were combined to include both sexes and OVX (representing female) and ORX terms (representing male), a bias toward reporting of female mice was evident. However, when the terms OVX and ORX were removed, a bias toward the use of male mice was identified. Thus, studies focusing on sex hormones are biased toward female reporting with all other studies biased in reporting of male mice. We now call upon journal editors to introduce consistent guidance for transparent and accessible reporting of murine sex in skeletal research to better monitor preclinical sex bias, to diversify development of treatments for bone health, and to enable global skeletal health equity. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper.

Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential.

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.