Murine Progeria Model Exhibits Delayed Fracture Healing with Dysregulated Local Immune Response.

Background: Bone fracture is one of the most globally prevalent injuries, with an estimated 189 million bone fractures occurring annually. Delayed union or nonunion occurs in up to 15% of fractures and involves the interruption or complete failure of bone continuity following fracture. Preclinical testing is essential to support the translation of novel strategies to promote improved fracture repair treatment, but there is a paucity of small animal models that recapitulate clinical attributes associated with delayed fracture healing. This study explores whether the Zmpste24 -/- (Z24 -/- ) knockout mouse model of Hutchinson-Gilford progeria syndrome presents with delayed fracture healing. Leveraging the previously characterized Z24 -/- phenotype of genomic instability, epigenetic changes, and fragility, we hypothesize that these underlying alterations will lead to significantly delayed fracture healing relative to age-matched wild type (WT) controls. Methods: WT and Z24 -/- mice received intramedullary fixed tibia fractures at ∼12 weeks of age. Mice were sacrificed throughout the time course of repair for the collection of organs that would provide information regarding the local (fracture callus, bone marrow, inguinal lymph nodes) versus peripheral (peripheral blood, contralateral tibia, abdominal organs) tissue microenvironments. Analyses of these specimens include histomorphometry, µCT, mechanical strength testing, protein quantification, gene expression analysis, flow cytometry for cellular senescence, and immunophenotyping. Results: Z24 -/- mice demonstrated a significantly delayed rate of healing compared to WT mice with consistently smaller fracture calli containing higher proportion of cartilage and less bone after injury. Cellular senescence and pro-inflammatory cytokines were elevated in the Z24 -/- mice before and after fracture. These mice further presented with a dysregulated immune system, exhibiting generally decreased lymphopoiesis and increased myelopoiesis locally in the bone marrow, with more naïve and less memory T cell but greater myeloid activation systemically in the peripheral blood. Surprisingly, the ipsilateral lymph nodes had increased T cell activation and other pro-inflammatory NK and myeloid cells, suggesting that elevated myeloid abundance and activation contributes to an injury-specific hyperactivation of T cells. Conclusion: Taken together, these data establish the Z24 -/- progeria mouse as a model of delayed fracture healing that exhibits decreased bone in the fracture callus, with weaker overall bone quality, immune dysregulation, and increased cellular senescence. Based on this mechanism for delayed healing, we propose this Z24 -/- progeria mouse model could be useful in testing novel therapeutics that could address delayed healing. The Translational Potential of this Article: This study employs a novel animal model for delayed fracture healing that researchers can use to screen fracture healing therapeutics to address the globally prevalent issue of aberrant fracture healing.

PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary mechanisms.

Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.

CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair.

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our In vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

CD47 is Required for Mesenchymal Progenitor Proliferation and Fracture Repair.

CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.

Dosage-dependent effects of FGFR2W290R mutation on craniofacial shape and cellular dynamics of the basicranial synchondroses.

Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.

Genetic architecture of trait variance in craniofacial morphology.

The genetic architecture of trait variance has long been of interest in genetics and evolution. One of the earliest attempts to understand this architecture was presented in Lerner's Genetic Homeostasis (1954). Lerner proposed that heterozygotes should be better able to tolerate environmental perturbations because of functional differences between the alleles at a given locus, with each allele optimal for slightly different environments. This greater robustness to environmental variance, he argued, would result in smaller trait variance for heterozygotes. The evidence for Lerner's hypothesis has been inconclusive. To address this question using modern genomic methods, we mapped loci associated with differences in trait variance (vQTL) on 1,101 individuals from the F34 of an advanced intercross between LG/J and SM/J mice. We also mapped epistatic interactions for these vQTL in order to understand the influence of epistasis for the architecture of trait variance. We did not find evidence supporting Lerner's hypothesis, that heterozygotes tend to have smaller trait variances than homozygotes. We further show that the effects of most mapped loci on trait variance are produced by epistasis affecting trait means and that those epistatic effects account for about a half of the differences in genotypic-specific trait variances. Finally, we propose a model where the different interactions between the additive and dominance effects of the vQTL and their epistatic partners can explain Lerner's original observations but can also be extended to include other conditions where heterozygotes are not the least variable genotype.