Hambright, W. S. | Search bioRxiv

bioRxiv2024

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

ABSTRACTO_ST_ABSBackgroundC_ST_ABSBone 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. MethodsWT 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, CT, mechanical strength testing, protein quantification, gene expression analysis, flow cytometry for cellular senescence, and immunophenotyping. ResultsZ24-/- 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 naive 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. ConclusionTaken 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 ArticleThis 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.

molecular biology↗

bioRxiv2022

Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells

Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence, in particular, is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine and chemokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration and repair. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potentetial of MSCs. To combat these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to human MSCs during the culture expansion process have not yet been elucidated. To address this, analyzed markers of senescence during culturing of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated {beta}-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs. Signficance StatementThe accumulation of dysfunctional, senescent cells throughout aging is not confined to specific tissues and cell types, but instead effects the whole body, including stem cells. Similarly, during culture expansion stem cells accumulate senescence while concurrently losing their regenerative potential. In this study, we found that fisetin (a well known senotherapeutic agent) can reduce the number of senescent cells during stem cell expansion. The current results indicate that fisetin may be used not only as a promising therapeutic to remove senescent cells in stem cell isolates from older individuals but also to reduce the accumulation of senescence during culture expansion.

cell biology↗