Biology subjects

Silva, M. J.

Publications and source records attributed to Silva, M. J..

11 recordsLinked to original sources

bioRxiv2026

Bone Response to Mechanical Loading is Highly Heritable and Correlates with Osteocyte Lacunar Morphology in Genetically Diverse Mouse Strains

Skeletal loading is a key regulator of bone formation. Studies using in vivo loading of rodent bones have established many mechanical and biological factors that regulate bone response to loading. However, most studies have been performed in a small number of inbred rodent strains, and there are few data on effects of genetic background on the loading response. In the current study we employed two murine models of genetic diversity, the Diversity Outbred (DO) and their eight founder inbred strains, to assess the influence of genetic background on skeletal response to tibial loading. Loading-induced periosteal bone formation rates and formed bone volumes varied significantly among inbred mouse strains. DO mice, which each carry a unique mixture of alleles of the inbred founder strains, were more responsive to loading, on average, than the the inbred founder strains. Additionally, bone response to mechanical loading was highly heritable; broad-sense heritability for measures of periosteal formation were near or above 80%. Furthermore, osteocyte lacunar morphology correlated with loading response across inbred strains. Inbred 129S1 mice had the most elongated lacunae and exhibited the highest magnitude of response on both periosteal and endocortical surfaces. 129S1 also demonstrated a robust transcriptional response to loading, although the pathways induced by loading were largely conserved with lab-standard C57Bl/6. Together, these findings demonstrate that genetic background significantly contributes to bone response to loading and establish DO mice as a platform for discovery of genes that regulate skeletal mechanoresponsiveness.

bioengineering↗

bioRxiv2026

Protocol for 3D digital dynamic histomorphometry of bone via time-lapse registration of serial microCT scans

Serial in vivo microCT enables quantification of dynamic bone remodeling by capturing formation and resorption over time. This protocol uses Dragonfly to register pre- and post-intervention scans of rodent long bones through an intuitive drag-and-click workflow that requires no coding expertise. Rigid registration enables spatial mapping and quantification of formed, resorbed, and quiescent cortical and trabecular bone at periosteal and endosteal surfaces across multiple tibial regions, providing a non-destructive and complementary alternative to classic, fluorochrome-based dynamic histomorphometry. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/728585v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e20e89org.highwire.dtl.DTLVardef@12872fcorg.highwire.dtl.DTLVardef@951694org.highwire.dtl.DTLVardef@1fd52f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Spatial transcriptomics for gene discovery identifies Slc13a5 as a modulator of bone mechanoadaptation

Bone is a dynamic tissue that continuously adapts its structure in response to mechanical loading, an essential process for maintaining skeletal health. However, this adaptive capacity declines with aging, contributing to increased fragility and fracture risk. Developing therapeutic strategies that preserve or restore bone mechanoadaptation in patients with increased bone fragility requires identifying key molecular regulators of this process. We applied spatial transcriptomics (GeoMx, NanoString) to characterize gene expression changes induced by mechanical loading in the murine tibia, focusing on periosteal and bone compartments in regions under tension and compression. Spatial data were validated and cross-compared with previously published bulk RNA-seq and laser-capture microdissection datasets, identifying a set of 12 genes consistently regulated by loading across independent platforms and laboratories. As part of a functional analysis, we selected Slc13a5, a citrate transporter implicated in bone mineralization and metabolism. Conditional deletion of Slc13a5 in osteolineage cells using Osteocalcin-Cre significantly increased the loading-induced mineralizing surface in tensile regions compared with Cre- Slc13a5fl/fl littermates. In addition, Slc13a5 cKO mice exhibited lower resorption around the neutral axis after loading compared to controls. Together, these findings identify Slc13a5 as a regulator of bone adaptation in regions experiencing low mechanical stimulation and suggest it as a potential therapeutic target for conditions characterized by impaired mechanoadaptive responses. This study highlights spatial transcriptomics as a powerful gene discovery framework for bone, enabling identification of novel targets to understand mechanisms and develop therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/711126v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14fc0e9org.highwire.dtl.DTLVardef@1023a16org.highwire.dtl.DTLVardef@133aa0eorg.highwire.dtl.DTLVardef@e19c86_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Combination Treatment with Sclerostin and Dkk1 Antibodies Synergizes with Tibial Loading to Stimulate Bone Formation in Aged Mice

Aging is associated with decreased bone formation and bone mass and increased fracture risk. Wnt pathway activation by mechanical loading is a potent strategy to improve bone mass, however, load-induced bone formation is diminished with aging. Neutralizing antibody (Ab) therapies targeting Wnt pathway inhibitors Sclerostin (Scl) and Dickkopf-related protein 1 (Dkk1) have proven successful in preclinical and clinical osteoporotic conditions. We asked whether treatment combining Scl-Ab and Dkk1-Ab can increase load-induced bone formation in a preclinical model of osteoporosis. Aged (22-month) C57BL/6N female mice underwent combination Scl-Ab plus Dkk1-Ab therapy (15 mg/kg each; subcutaneously; 2x/wk; saline control) for 2 weeks, concomitant with a mechanical loading regimen previously shown to induce modest bone formation in tibias of aged mice (-2200 {varepsilon}, 1200 cycles/day, 5 day/wk). Changes in bone morphology and formation were assessed by longitudinal microCT and dynamic histomorphometry, respectively. Molecular indices of bone formation and Wnt pathway activation were assessed by qPCR of cortical bone. Treatment with Scl-Ab plus Dkk1-Ab induced significant improvements in cancellous (BV/TV +50%) and cortical morphology (Ct.Th +25%) in non-loaded limbs of antibody-treated mice vs. vehicle control mice. Importantly, periosteal bone formation rate was 10-fold higher in loaded limbs of antibody versus vehicle treated mice, indicating a synergistic effect. Gene expression analysis showed that antibody treatment and loading synergistically upregulated Wnt1 expression, which may have contributed to the observed synergistic effect on bone formation. These results confirm the potent anabolic effect of combination Scl plus Dkk1 antibody treatment. Moreover, they show that antibody treatment and skeletal loading are more effective at increasing periosteal bone formation in aged mice than either treatment alone. These findings support the concept that combinatorial therapy using dual Scl and Dkk1 antibodies plus weight-bearing exercise may be an effective treatment for age-related osteoporosis.

physiology↗

bioRxiv2025

Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

Fractures heal by rapid formation of mineralized callus. Essential to this process is proliferation of periosteal cells to supply bone-forming osteoblasts. To better understand the role of cell proliferation in fracture healing, we asked: How is callus composition altered when osteolineage cells proliferation is blocked? Do mature osteoblasts proliferate to contribute to callus formation? First, mice expressing herpes simplex virus-thymidine kinase (HSV-TK) driven by the 3.6Col1a1 promoter were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Analysis of callus cells using single-cell RNA-seq revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than control mice, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells that expressed the early osteoblast markers osterix (OSX) and the late marker osteocalcin (OCN) were in the cell cycle. Next, we targeted proliferating osteoblasts at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with novel ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited poorer radiographic healing, decreased callus bone volume and a shift from callus bone to fibrous tissue. We conclude that osteoblasts, often considered post-mitotic, proliferate after fracture to contribute to formation of mineralized callus essential to healing.

physiology↗

bioRxiv2024

Spatial histomorphometry reveals that local peripheral nerves modulate but are not required for skeletal adaptation to applied load in mice

Mechanical loading is required for bone health and results in skeletal adaptation to optimize strength. Local nerve axons, particularly within the periosteum, may respond to load-induced biomechanical and biochemical cues. However, their role in the bone anabolic response remains controversial. We hypothesized that spatial alignment of periosteal nerves with sites of load-induced bone formation would clarify this relationship. To achieve this, we developed RadialQuant, a custom tool for spatial histomorphometry. Tibiae of control and neurectomized (sciatic/femoral nerve cut) pan-neuronal Baf53b-tdTomato reporter mice were loaded for 5-days. Bone formation and periosteal nerve axon density were then quantified simultaneously in non-decalcified sections of the mid-diaphysis using RadialQuant. In control animals, anabolic loading induced maximal periosteal bone formation at the site of peak compression, as has been reported previously. Loading did not significantly change overall periosteal nerve density. However, a trending 28% increase in periosteal axons was noted at the site of peak compression in loaded limbs. Neurectomy depleted 88% of all periosteal axons, with near-total depletion on load-responsive surfaces. Neurectomy alone also caused de novo bone formation on the lateral aspect of the mid-diaphysis. However, neurectomy did not inhibit load-induced increases in periosteal bone area, mineralizing surface, or bone formation rate. Rather, neurectomy spatially redistributed load-induced bone formation towards the lateral tibial surface with a reduction in periosteal bone formation at the posterolateral apex (-63%) and enhancement at the lateral surface (+1360%). Altogether, this contributed to comparable load-induced changes in cortical bone area fraction (+4.4% in controls; +5.4% in neurectomized). Our results show that local skeletal innervation modulates but is not required for skeletal adaptation to applied load. This supports the continued use of loading and weight-bearing exercise as an effective strategy to increase bone mass, even in patients with peripheral nerve damage or dysfunction. LAY SUMMARYMechanical loading is required for bone health and can increase new bone formation to optimize strength and reduce fractures. Increased bone formation with loading is primarily mediated by osteocytes, a specialized cell type that is embedded throughout the bone matrix. Local nerve axons, particularly within the periosteum, may also respond to load-induced biomechanical and biochemical cues and could contribute to new bone formation in response to load. However, their role in the bone anabolic response remains controversial. To address this, we studied the adaptation of bone to applied load in the presence of local periosteal nerves, and in settings where the nerves had been surgically removed. Our results show that local skeletal innervation modulates but is not required for skeletal adaptation to applied load. This supports the continued use of loading and weight-bearing exercise as an effective strategy to increase bone mass, even in patients with peripheral nerve damage or dysfunction.

physiology↗

bioRxiv2023

Multi-Scale Cortical Bone Traits Vary in Two Mouse Models of Genetic Diversity

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that cant easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of three long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from two populations of genetic diversity. Additionally, we compared how intra-bone relationships varied in the two populations. Our first population of genetic diversity included 72 females and 72 males from the eight Inbred Founder strains used to create the Diversity Outbred (DO) population. These eight strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the two populations of genetic diversity, we show each DO mouse does not resemble a single Inbred Founder but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intra-bone relationships (e.g., ultimate force vs. cortical area) were mainly conserved in our two populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/543484v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1e61c1org.highwire.dtl.DTLVardef@a3f941org.highwire.dtl.DTLVardef@1411dceorg.highwire.dtl.DTLVardef@bfc8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2021

Multi-trait and multi-environment Bayesian analysis to predict the G x E interaction in flood-irrigated rice

The biggest challenge for the reproduction of flood-irrigated rice is to identify superior genotypes that present development of high-yielding varieties with specific grain qualities, resistance to abiotic and biotic stresses in addition to superior adaptation to the target environment.Thus, the objectives of this study were to propose a multi-trait and multi-environment Bayesian model to estimate genetic parameters for the flood-irrigated rice crop. To this end, twenty-five rice genotypes belonging to the flood-irrigated rice breeding program were evaluated. Grain yield and flowering were evaluated in the agricultural year 2017/2018. The experimental design used in all experiments was a randomized block design with three replications. The Markov Chain Monte Carlo algorithm was used to estimate genetic parameters and genetic values. The flowering is highly heritable by the Bayesian credibility interval: h2 = 0.039-0.80, and 0.02-0.91, environment 1 and 2, respectively. The genetic correlation between traits was significantly different from zero in the two environments (environment 1: -0.80 to 0.74; environment 2: -0.82 to 0.86. The relationship of CVe and CVg higher for flowering in the reduced model (CVg/CVe= 5.83 and 13.98, environments 1 and 2, respectively). For the complete model, this trait presented an estimate of the relative variation index of: CVe= 4.28 and 4.21, environments 1 and 2, respectively. In summary, the multi-trait and multi-environment Bayesian model allowed a reliable estimate of the genetic parameter of flood-irrigated rice. Bayesian analyzes provide robust inference of genetic parameters. Therefore, we recommend this model for genetic evaluation of flood-irrigated rice genotypes, and their generalization, in other crops. Precise estimates of genetic parameters bring new perspectives on the application of Bayesian methods to solve modeling problems in the genetic improvement of flood-irrigated rice.

plant biology↗

bioRxiv2021

Osteoblast-Specific Wnt Secretion is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in adult bone homeostasis and the anabolic response to mechanical loading is not well documented. We inhibited Wnt ligand secretion in adult (5-mo) mice using a systemic (drug) and a bone-targeted (genetic) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the porcupine inhibitor WNT974 exhibited a decrease in bone formation in non-loaded limbs as well as a 54% decline in the periosteal bone formation response to tibial loading. Similarly, within 1-2 weeks of Wls deletion in osteoblasts (Osx-CreERT2;WlsF/F mice), skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (WlsF/F). These findings establish a requirement for Wnt ligand secretion by osteoblasts for adult bone homeostasis and the anabolic response to mechanical loading.

genetics↗

bioRxiv2020

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks post fracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and microCT scans, had delayed bone bridging and reduced callus size. Following sacrifice, ex vivo microCT and histology demonstrated failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing demonstrated a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion.

bioengineering↗

bioRxiv2018

Osteocyte death and bone overgrowth in mice lacking Fibroblast Growth Factor Receptors 1 and 2 in mature osteoblasts and osteocytes

Fibroblast Growth Factor (FGF) signaling pathways have well established roles in skeletal development, with essential functions in both chondrogenesis and osteogenesis. In mice, previous conditional knockout studies suggested distinct roles for FGF receptor 1 (FGFR1) signaling at different stages of osteogenesis and a role for FGFR2 in osteoblast maturation. However, the potential for redundancy among FGFRs and the mechanisms and consequences of stage-specific osteoblast lineage regulation were not addressed. Here, we conditionally inactivate Fgfr1 and Fgfr2 in mature osteoblasts with an Osteocalcin-Cre or Dentin matrix protein 1-CreER driver. We find that young mice lacking both receptors or only FGFR1 are phenotypically normal. However, after 6 weeks of age these Fgfr1/Fgfr2 double- and Fgfr1 single-conditional knockout mice develop a high bone mass phenotype with increased periosteal apposition, increased endocortical woven bone with increased porosity, and biomechanical properties that reflect increased bone mass but impaired material properties. Histopathological and gene expression analyses show that this phenotype is preceded by a striking loss of osteocytes, and gradual activation of the Wnt/{beta}Catenin signaling pathway. These data identify a role for FGFR1 signaling in mature osteoblasts/osteocytes that is required for osteocyte survival during postnatal bone growth.

developmental biology↗