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.

Multi-scale cortical bone traits vary in females and males from 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 cannot 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 3 long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from 2 populations of genetic diversity. Additionally, we compared how intrabone relationships varied in the 2 populations. Our first population of genetic diversity included 72 females and 72 males from the 8 inbred founder strains used to create the Diversity Outbred (DO) population. These 8 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 2 populations of genetic diversity, we show that 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, intrabone relationships (eg, ultimate force vs. cortical area) were mainly conserved in our 2 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.

Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Cyclic testing of six-strand suture techniques for zone 2 flexor tendon lacerations.

BACKGROUND: Biomechanical analysis using cyclic testing for repaired flexor tendons is a clinically relevant method. The aim of this study was to evaluate the tensile properties of two six-strand suture techniques, the triple looped suture and Yoshizu #1 suture techniques using cyclic testing under simulating early active mobilization conditions. METHODS: Twenty-five flexor digitorum profundus tendons harvested from fresh frozen human cadaver hands were repaired in zone 2 utilizing one of three repair techniques: the 2-strand modified Kessler (MK) technique as a control, the triple looped suture (TLS) and Yoshizu #1 suture (Y1) techniques. In each suture technique, 4-0 monofilament nylon sutures were used for core sutures and 6-0 monofilament nylon sutures for circumferential running sutures. Cyclic testing was performed using 20 N with 600 cycles at 1 Hz. RESULTS: Five out of eight specimens in the MK group ruptured during cyclic testing. Thus, this group was excluded from analysis. On the other hand, all tendons in the TLS and Y1 groups tolerated cyclic testing. Average gaps of the TLS and Y1 groups were 0.5 ± 0.8 mm and 1.9 ± 2.2 mm, respectively. All tendons in the TLS group and six out of nine tendons in the Y1 group formed gaps less than 2 mm. Two tendons in the Y1 group formed a gap of 3.8 and 6.6 mm had breakage of peripheral sutures at the first cycle. Mean ultimate tensile force of the TLS and Y1 group measured after cyclic tensing, were 66.2 ± 9.0 N and 65.9 ± 13.1 N, respectively. No statistical difference between the two groups was found in gap and ultimate tensile forces. CONCLUSIONS: This study suggested that the TLS and Y1 techniques have tensile properties to allow early active mobilization. None of tendons repaired with the TLS technique had gaps more than 2 mm.

Mitapivat: A Quinolone Sulfonamide to Manage Hemolytic Anemia in Adults With Pyruvate Kinase Deficiency.

BACKGROUND: Pyruvate kinase (PK) deficiency is a rare enzyme-linked glycolytic defect resulting in mild-to-severe chronic persistent erythrocyte hemolysis. The disease is an autosomal recessive trait caused by mutations in the PK liver and red blood cell gene characterized by insufficient erythrocyte PK activity. PK deficiency is most diagnosed in persons of northern European descent and managed with packed red blood cell transfusions, chelation, and splenectomy with cholecystectomy. Mitapivat is the first approved therapy indicated for hemolytic anemia in adults with PK deficiency with the potential for delaying splenectomy in mild-moderate disease. MECHANISM OF ACTION, PHARMACODYNAMICS, AND PHARMACOKINETICS: Mitapivat is a PK activator that acts by allosterically binding to the PK tetramer and increases PK activity. The red blood cell form of PK is mutated in PK deficiency, which leads to reduced adenosine triphosphate, shortened red blood cell lifespan, and chronic hemolysis. The half-life of elimination is 3-5 hours, with 73% bioavailability, 98% plasma protein binding, and a median duration of response of 7 months. CLINICAL TRIALS: Mitapivat has been investigated through various clinical trials for different therapeutic indications. Pivotal trials that serve the primary focus throughout this article are ACTIVATE, ACTIVATE-T, and RISE. ACTIVATE is a phase 3, randomized, double-blind, placebo-controlled study that evaluated the efficacy and safety of mitapivat in adult patients who were not receiving regular blood transfusions. Contrarily, ACTIVATE-T explored the safety and efficacy of mitapivat in adults with PK deficiency who received regular blood transfusions. Both trials demonstrated favorable use of mitapivat in PK deficiency. Focusing on another indication, the ongoing RISE trial investigates the optimal dosage of mitapivat in sickle cell disease. THERAPEUTIC ADVANCE: Mitapivat is an appropriate treatment for adults with PK deficiency requiring transfusions and may be considered for patients with symptomatic anemia who do not require transfusions and/or PK deficiency with compensated hemolysis without overt anemia.

Pharmacist telehealth in an underserved urban population with type 2 diabetes mellitus.

BACKGROUND: During the pre-vaccine months of the COVID-19 pandemic, pharmacists providing comprehensive medication management to underserved patients with type 2 diabetes mellitus at an urban Federally Qualified Healthcare Center shifted to telephone-based telehealth. OBJECTIVES: This retrospective, observational cohort study evaluated the effectiveness of clinical pharmacist telehealth while identifying associations between patient characteristics and efficacy measures. METHODS: Patients with uncontrolled type 2 diabetes (hemoglobin A1c (HbA1c) ≥ 8%) with a clinical pharmacist visit between April 1 and August 31, 2020, were included. Telehealth effectiveness was measured by the proportions of: 1) patients reached, 2) appointments completed, and 3) the median change in HbA1c from baseline. Interventions by the clinical pharmacist were analyzed as a secondary outcome. RESULTS: There were 181 patients scheduled and 172 (95%) of those patients kept at least one appointment. Of the 667 appointments scheduled, 73% were kept. Median HbA1c was reduced from 10.2% to 9.2% over 5 months of follow-up, and 24.6% of patients achieved a HbA1c < 8% (n = 138, p < 0.0001 for each). Greater HbA1c changes were associated with higher baseline blood glucose (p = 0.01), higher baseline HbA1c (p < 0.0001), non-insulin medications at baseline (p = 0.007) and among those with more kept visits (p = 0.03). The healthcare quality impact of interventions during each appointment was favorable; 83.3% brought care to a higher standard, 1.9% averted major organ dysfunction and 0.4% prevented death. CONCLUSIONS: Clinical pharmacist telehealth was effective for providing patient-centered diabetes care when in-person office visits were not an option.

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 can't 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.

T cells heal bone fractures with help from the gut microbiome.

Immune cells play an important functional role in bone fracture healing. Fracture repair is a well-choreographed process that takes approximately 21 days in healthy mice. While the process is complex, conceptually it can be divided into four overlapping stages: inflammation, cartilaginous callus formation, bony callus formation, and remodeling. T cells play a key role in both the cartilaginous and bony callus phases by producing IL-17A. In this issue of the JCI, Dar et al. showed that T cells were recruited from the gut, where the gut microbiota determined the pool of T cells that expressed IL-17A. Treatment with antibiotics and dysbiosis reduced the expansion of IL-17-expressing CD4+ T cells (Th17) and impaired callus formation. These findings demonstrate crosstalk among the gut microbiota, the adaptive immune system, and bone that has clinical implications for fracture healing.

Cortical bone relationships are maintained regardless of sex and diet in a large population of LGXSM advanced intercross mice.

Introduction: Knowledge of bone structure-function relationships in mice has been based on relatively small sample sets that limit generalizability. We sought to investigate structure-function relationships of long bones from a large population of genetically diverse mice. Therefore, we analyzed previously published data from the femur and radius of male and female mice from the F34 generation of the Large-by-Small advanced intercross line (LGXSM AI), which have over a two-fold continuous spread of bone and body sizes (Silva et al. 2019 JBMR). Methods: Morphological traits, mechanical properties, and estimated material properties were collected from the femur and radius from 1113 LGXSM AI adult mice (avg. age 25 wks). Males and females fed a low-fat or high-fat diet were evaluated to increase population variation. The data were analyzed using principal component analysis (PCA), Pearson's correlation, and multivariate linear regression. Results: Using PCA groupings and hierarchical clustering, we identified a reduced set of traits that span the population variation and are relatively independent of each other. These include three morphometry parameters (cortical area, medullary area, and length), two mechanical properties (ultimate force and post-yield displacement), and one material property (ultimate stress). When comparing traits of the femur to the radius, morphological traits are moderately well correlated (r2: 0.18-0.44) and independent of sex and diet. However, mechanical and material properties are weakly correlated or uncorrelated between the long bones. Ultimate force can be predicted from morphology with moderate accuracy for both long bones independent of variations due to genetics, sex, or diet; however, predictions miss up to 50 % of the variation in the population. Estimated material properties in the femur are moderately to strongly correlated with bone size parameters, while these correlations are very weak in the radius. Discussion: Our results indicate that variation in cortical bone phenotype in the F34 LGXSM AI mouse population can be adequately described by a reduced set of bone traits. These traits include cortical area, medullary area, bone length, ultimate force, post-yield displacement, and ultimate stress. The weak correlation of mechanical and material properties between the femur and radius indicates that the results from routine three-point bending tests of one long bone (e.g., femur) may not be generalizable to another long bone (e.g., radius). Additionally, these properties could not be fully predicted from bone morphology alone, confirming the importance of mechanical testing. Finally, material properties of the femur estimated based on beam theory equations showed a strong dependence on geometry that was not seen in the radius, suggesting that differences in femur size within a study may confound interpretation of estimated material properties.

Loading-induced bone formation is mediated by Wnt1 induction in osteoblast-lineage cells.

Mechanical loading on the skeleton stimulates bone formation. Although the exact mechanism underlying this process remains unknown, a growing body of evidence indicates that the Wnt signaling pathway is necessary for the skeletal response to loading. Recently, we showed that Wnts produced by osteoblast lineage cells mediate the osteo-anabolic response to tibial loading in adult mice. Here, we report that Wnt1 specifically plays a crucial role in mediating the mechano-adaptive response to loading. Independent of loading, short-term loss of Wnt1 in the Osx-lineage resulted in a decreased cortical bone area in the tibias of 5-month-old mice. In females, strain-matched loading enhanced periosteal bone formation in Wnt1F/F controls, but not in Wnt1F/F; OsxCreERT2 knockouts. In males, strain-matched loading increased periosteal bone formation in both control and knockout mice; however, the periosteal relative bone formation rate was 65% lower in Wnt1 knockouts versus controls. Together, these findings show that Wnt1 supports adult bone homeostasis and mediates the bone anabolic response to mechanical loading.

VEGFA from osteoblasts is not required for lamellar bone formation following tibial loading.

The relationship between osteogenesis and angiogenesis is complex. Normal bone development requires angiogenesis, mediated by vascular endothelial growth factor A (VEGFA). Studies have demonstrated through systemic inhibition or genetic modification that VEGFA is indispensable for several types of bone repair, presumably via its role in supporting angiogenesis. But a direct role for VEGFA within osteoblasts, in the absence of angiogenesis, has also been suggested. To address the question of whether VEGFA from osteoblasts supports bone formation directly, we applied anabolic loading to induce lamellar bone formation in mice, a process shown to be independent of angiogenesis. We hypothesized that VEGFA from osteoblasts is required for lamellar bone formation. To test this hypothesis, we applied axial tibial compression to inducible Cre/LoxP mice from three lines. Vegfafl/fl mice were crossed with Ubiquitin C (UBC), Osterix (Osx) and Dentin-Matrix Protein 1 (DMP1) Cre-ERT2 mice to target all cells, (pre)osteoblast-lineage cells, and mature osteoblasts and osteocytes, respectively. Genotype effects were determined by comparing control (Vegfafl/fl) and Cre+ (VegfaΔ) mice for each line. At 5 months of age tamoxifen was injected for 5 days followed by a 3-week clearance prior to loading. Female and male mice (N = 100) were loaded for 5 days to peak forces to engender -3100 µÎµ peak compressive strain and processed for dynamic histomorphometry (day 12). Percent MS/BS increased 20-70 % as a result of loading, with no effect of genotype in Osx or Dmp1 lines. In contrast, the UBC groups had a significant decrease in relative periosteal BFR/BS in VegfaΔ vs. Vegfafl/fl mice. The UBC line did not have any cortical bone phenotype in non-loaded femurs. In summary, dynamic histomorphometry data confirmed that tibial loading induces lamellar bone formation. Contrary to our hypothesis, there was no decrease in loading-induced bone formation in the Osx or Dmp1 lines in the absence of VEGFA. There was a decrease in bone formation in the UBC line where all cells were targeted. This result indicates that VEGFA from a non-osteoblast cell source supports loading-induced lamellar bone formation, although osteoblast/osteocyte VEGFA is dispensable. These findings support a paracrine model whereby non-osteoblast VEGFA supports lamellar bone formation, independent of angiogenesis.

Cryogel Scaffold-Mediated Delivery of Adipose-Derived Stem Cells Promotes Healing in Murine Model of Atrophic Non-Union.

Non-union is defined as the permanent failure of a bone to heal and occurs clinically in 5% of fractures. Atrophic non-unions, characterized by absent/minimal callus formation, are poorly understood and difficult to treat. We recently demonstrated a novel murine model of atrophic non-union in the 3.6Col1A1-tk (Col1-tk) mouse, wherein dosing with the nucleoside analog ganciclovir (GCV) was used to deplete proliferating osteoprogenitor cells, leading to a radiographic and biomechanical non-union after the mid-shaft femur fracture. Using this Col1-tk atrophic non-union model, we hypothesized that the scaffold-mediated lentiviral delivery of doxycycline-inducible BMP-2 transgenes would induce osteogenesis at the fracture site. Cryogel scaffolds were used as a vehicle for GFP+ and BMP-2+ cell delivery to the site of non-union. Cryogel scaffolds were biofabricated through the cross-linking of a chitosan-gelatin polymer solution at subzero temperatures, which results in a macroporous, spongy structure that may be advantageous for a bone regeneration application. Murine adipose-derived stem cells were seeded onto the cryogel scaffolds, where they underwent lentiviral transduction. Following the establishment of atrophic non-unions in the femurs of Col1-tk mice (4 weeks post-fracture), transduced, seeded scaffolds were surgically placed around the site of non-union, and the animals were given doxycycline water to induce BMP-2 production. Controls included GFP+ cells on the cryogel scaffolds, acellular scaffolds, and sham (no scaffold). Weekly radiographs were taken, and endpoint analysis included micro-CT and histological staining. After 2 weeks of implantation, the BMP-2+ scaffolds were infiltrated with cartilage and woven bone at the non-union site, while GFP+ scaffolds had woven bone formation. Later, timepoints of 8 weeks had woven bone and vessel formation within the BMP-2+ and GFP + scaffolds with cortical bridging of the original fracture site in both groups. Overall, the cell-seeded cryogels promoted osseous healing. However, while the addition of BMP-2 promoted the endochondral ossification, it may provide a slower route to healing. This proof-of-concept study demonstrates the potential for cellularized cryogel scaffolds to enhance the healing of non-unions.

Fracture healing is delayed in the absence of gasdermin-interleukin-1 signaling.

Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1ß (IL-1ß) and IL-18. Excessive GSDM-mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1ß and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

Dmp1 Lineage Cells Contribute Significantly to Periosteal Lamellar Bone Formation Induced by Mechanical Loading But Are Depleted from the Bone Surface During Rapid Bone Formation.

Previous work has shown that osteoprogenitor cells (Prx1+) and pre-osteoblasts (Osx+) contribute to mechanical loading-induced bone formation. However, the role of mature Dmp1-expressing osteoblasts has not been reported. In this study we assessed the contribution of osteoblast lineage cells to bone formation at an early time point following mechanical loading (day 8 from onset of loading). We labeled Osx-expressing and Dmp1-expressing cells in inducible Osx and Dmp1 reporter mice (iOsx-Ai9, iDmp1-Ai9), respectively, 3 weeks before loading. Mice were then loaded daily for 5 days (days 1-5) and were dosed with 5-ethynyl-2'-deoxyuridine (EdU) in their drinking water until euthanasia on day 8. Mice were loaded to lamellar and woven bone inducing stimulation (-7 N/1400 µÎµ, -10 N/2000 µÎµ) to assess differences in these processes. We found varied responses in males and females to the loading stimuli, inducing modest lamellar (females, -7 N), moderate lamellar (males, -10 N), and robust woven (females, -10 N) bone. Overall, we found that preexisting (ie, lineage positive) Osx-expressing and Dmp1-expressing cells contribute largely to the bone formation response, especially during modest bone formation, while our results stuggest that other (non-lineage-positive) cells support the sustained bone formation response during rapid bone formation. With moderate or robust levels of bone formation, a decrease in preexisting Osx-expressing and Dmp1-expressing cells at the bone surface occurred, with a near depletion of Dmp1-expressing cells from the surface in female mice loaded to -10 N (from 52% to 11%). These cells appeared to be replaced by lineage-negative cells from the periosteum. We also found a dose response in proliferation, with 17% to 18% of bone surface cells arising via proliferation in modest lamellar, 38% to 53% in moderate lamellar, and 59% to 81% in robust woven bone formation. In summary, our results show predominant contributions by preexisting Osx and Dmp1 lineage cells to loading-induced lamellar bone formation, whereas recruitment of earlier osteoprogenitors and increased cell proliferation support robust woven bone formation. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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 the bone anabolic response to mechanical loading is not well established. Recent transcriptome profiling studies by our laboratory and others show that mechanical loading potently induces genes encoding Wnt ligands, including Wnt1 and Wnt7b. Based on these findings, we hypothesized that mechanical loading stimulates adult bone formation by inducing Wnt ligand expression. To test this hypothesis, we inhibited Wnt ligand secretion in adult (5 months old) mice using a systemic (drug) and a bone-targeted (conditional gene knockout) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the Wnt secretion inhibitor WNT974 exhibited a decrease in bone formation in non-loaded bones as well as a 54% decline in the periosteal bone formation response to tibial loading. Next, osteoblast-specific Wnt secretion was inhibited by dosing 5-month-old Osx-CreERT2; WlsF/F mice with tamoxifen. Within 1 to 2 weeks of Wls deletion, 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 ). Together, these findings show that Wnt ligand secretion is required for adult bone homeostasis, and furthermore establish a role for osteoblast-derived Wnts in mediating the bone anabolic response to tibial loading. © 2021 American Society for Bone and Mineral Research (ASBMR).

KIF26B Silencing Prevents Osseous Transdifferentiation of Progenitor/Stem Cells and Attenuates Ectopic Calcification in a Murine Model.

Ectopic calcification is an osteogenic process that leads to the formation of inappropriate bone within intra-articular soft tissues, often in response to injury or surgery. The molecular mechanisms governing this phenotype have yet to be determined. Using a population genetics approach, we identified an association of the kinesin superfamily member 26b (Kif26b) with injury-induced ectopic calcification through quantitative trait locus analysis of recombinant inbred mouse strains, consistent with a genomewide association study that identified KIF26B as a severity locus for ectopic calcification in patients with hip osteoarthritis. Despite these associations of KIF26B with ectopic calcification, its mechanistic role and functional implications have not yet been fully elucidated. Here, we aim to decipher the functional role of KIF26B in osseous and chondrogenic transdifferentiation of human and murine progenitor/stem cells and in a murine model of non-invasive injury-induced intra-articular ectopic calcification. We found that KIF26B ablation via lentivirus-mediated shRNA significantly arrested osteogenesis of progenitor/stem cells and suppressed the expression of typical osteogenic marker genes. Conversely, KIF26B loss-of-function increased chondrogenesis as demonstrated by enhanced Safranin-O staining and by the elevated expression of chondrogenic marker genes. Furthermore, cell function analysis revealed that KIF26B knockdown significantly decreased cell viability and proliferation and induced cellular apoptosis. Mechanistically, loss of osteogenesis was reverted by the addition of a Wnt agonist, SKL2001, demonstrating a role of KIF26B in canonical Wnt/ß-catenin signaling. Finally, intra-articular delivery of Kif26b shRNA in B6-129SF2/J mice significantly hampered the development of intra-articular ectopic calcification at 8 weeks after injury compared with mice treated with non-target scrambled shRNA. In summary, these observations highlight that KIF26B plays a crucial role in ectopic bone formation by repressing osteogenesis, but not chondrogenesis, potentially via modulating Wnt/ß-catenin signaling. These findings establish KIF26B as a critical determinant of the osteogenic process in pathologic endochondral bone formation and an actionable target for pharmacotherapy to mitigate ectopic calcification (and heterotopic ossification). © 2021 American Society for Bone and Mineral Research (ASBMR).

Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

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 postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed 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. © 2021 American Society for Bone and Mineral Research (ASBMR).

Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes.

Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.

Type 1 diabetic Akita mice have low bone mass and impaired fracture healing.

Type 1 diabetes (T1DM) impairs bone formation and fracture healing in humans. Akita mice carry a mutation in one allele of the insulin-2 (Ins2) gene, which leads to pancreatic beta cell dysfunction and hyperglycemia by 5-6 weeks age. We hypothesized that T1DM in Akita mice is associated with decreased bone mass, weaker bones, and impaired fracture healing. Ins2 ± (Akita) and wildtype (WT) males were subjected to femur fracture at 18-weeks age and healing assessed 3-21 days post-fracture. Non-fractured left femurs were assessed for morphology (microCT) and strength (bending or torsion) at 19-21 weeks age. Fractured right femurs were assessed for callus mechanics (torsion), morphology and composition (microCT and histology) and gene expression (qPCR). Both Akita and WT mice gained weight from 3 to 18 weeks age, but Akita mice weighed less starting at 5 weeks (-5.2%, p < 0.05). At 18-20 weeks age Akita mice had reduced serum osteocalcin (-30%), cortical bone area (-16%), and thickness (-17%) compared to WT, as well as reduced cancellous BV/TV (-39%), trabecular thickness (-23%) and vBMD (-31%). Mechanical testing of non-fractured femurs showed decreased structural (stiffness, ultimate load) and material (ultimate stress) properties of Akita bones. At 14 and 21 days post fracture Akita mice had a significantly smaller callus than WT mice (~30%), with less cartilage and bone area. Assessment of torsional strength showed a weaker callus in Akita mice with lower stiffness (-42%), maximum torque (-44%) and work to fracture (-44%). In summary, cortical and cancellous bone mass were reduced in Akita mice, with lower bone mechanical properties. Fracture healing in Akita mice was impaired by T1DM, with a smaller, weaker fracture callus due to decreased cartilage and bone formation. In conclusion, the Akita mouse mimics some of the skeletal features of T1DM in humans, including osteopenia and impaired fracture healing, and may be useful to test interventions.