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Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding β-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated β-cateninGOF mRNA encapsulated in SM-102 LNPs (SM-102-β-cateninGOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2. When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 μg concentration at 2 weeks post-fracture. μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo, revealing significantly more bone volume over total volume in the 45 μg group. Thus, we generated a novel mRNA-based therapeutic encoding a β-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.

Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro. When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1β and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo. Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications.

ObjectiveTo evaluate clinical characteristics, outcome, and prognostic variables in a cohort of dogs surviving > 1 year after an initial diagnosis of osteosarcoma.DesignRetrospective case series.Animals90 client-owned dogs.ProceduresMedical records for an 11-year period from 1997 through 2008 were reviewed, and patients with appendicular osteosarcoma that lived > 1 year after initial histopathologic diagnosis were studied. Variables including signalment, weight, serum alkaline phosphatase activity, tumor location, surgery, and adjuvant therapies were recorded. Median survival times were calculated by means of a Kaplan-Meier survival function. Univariate analysis was conducted to compare the survival function for categorical variables, and the Cox proportional hazard model was used to evaluate the likelihood of death > 1 year after diagnosis on the basis of the selected risk factors.Results90 dogs met the inclusion criteria; clinical laboratory information was not available in all cases. Median age was 8.2 years (range, 2.7 to 13.3 years), and median weight was 38 kg (83.6 lb; range, 21 to 80 kg [46.2 to 176 lb]). Serum alkaline phosphatase activity was high in 29 of 60 (48%) dogs. The most common tumor location was the distal portion of the radius (54/90 [60%]). Eighty-nine of 90 (99%) dogs underwent surgery, and 78 (87%) received chemotherapy. Overall, 49 of 90 (54%) dogs developed metastatic disease. The median survival time beyond 1 year was 243 days (range, 1 to 1,899 days). Dogs that developed a surgical-site infection after limb-sparing surgery had a significantly improved prognosis > 1 year after osteosarcoma diagnosis, compared with dogs that did not develop infections.Conclusions and clinical relevanceResults of the present study indicated that dogs with an initial diagnosis of osteosarcoma that lived > 1 year had a median survival time beyond the initial year of approximately 8 months. As reported previously, the development of a surgical-site infection in dogs undergoing a limb-sparing surgery significantly affected prognosis and warrants further study.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro‐RNA (miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin‐filament‐based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells. [ABSTRACT FROM AUTHOR]

Abstract Background Though used for over a century, structural bone allografts suffer from a high rate of mechanical failure due to limited graft revitalization even after extended periods in vivo. Novel strategies that aim to improve graft incorporation are lacking but necessary to improve the long-term clinical outcome of patients receiving bone allografts. The current study evaluated the effect of low-intensity pulsed ultrasound (LIPUS), a potent exogenous biophysical stimulus used clinically to accelerate the course of fresh fracture healing, and longitudinal allograft perforations (LAP) as non-invasive therapies to improve revitalization of intercalary allografts in a sheep model. Methods Fifteen skeletally-mature ewes were assigned to five experimental groups based on allograft type and treatment: +CTL, -CTL, LIPUS, LAP, LIPUS+LAP. The +CTL animals (n = 3) received a tibial ostectomy with immediate replacement of the resected autologous graft. The -CTL group (n = 3) received fresh frozen ovine tibial allografts. The +CTL and -CTL groups did not receive LAP or LIPUS treatments. The LIPUS treatment group (n = 3), following grafting with fresh frozen ovine tibial allografts, received ultrasound stimulation for 20 minutes/day, 5 days/week, for the duration of the healing period. The LAP treatment group (n = 3) received fresh frozen ovine allografts with 500 μm longitudinal perforations that extended 10 mm into the graft. The LIPUS+LAP treatment group (n = 3) received both LIPUS and LAP interventions. All animals were humanely euthanized four months following graft transplantation for biomechanical and histological analysis. Results After four months of healing, daily LIPUS stimulation of the host-allograft junctions, alone or in combination with LAP, resulted in 30% increases in reconstruction stiffness, paralleled by significant increases (p < 0.001) in callus maturity and periosteal bridging across the host/allograft interfaces. Longitudinal perforations extending 10 mm into the proximal and distal endplates filled to varying degrees with new appositional bone and significantly accelerated revitalization of the allografts compared to controls. Conclusion The current study has demonstrated in a large animal model the potential of both LIPUS and LAP therapy to improve the degree of allograft incorporation. LAP may provide an option for increasing porosity, and thus potential in vivo osseous apposition and revitalization, without adversely affecting the structural integrity of the graft.

Appendicular osteosarcoma (OSA) remains a prevalent musculoskeletal cancer in dogs and definitive local control followed by adjuvant cytotoxic chemotherapy is considered the gold standard approach. Several studies support surgical limb salvage as a means of local control with similar outcomes compared with limb amputation. Complications are well described for limb salvage but little is known of dogs that undergo secondary amputation as a result of complications and outcomes specific to this group. A retrospective analysis of dogs in an institutional primary bone tumour registry was performed to identify dogs diagnosed with histologically confirmed OSA treated with surgical limb salvage with a technique that required an implant to reconstruct the osseous defect. A total of 192 dogs were identified with 31 dogs undergoing secondary amputation representing a limb preservation rate of 84%. A total of 111 dogs were analysed: 31 secondary amputation cases and 80 controls were selected for comparison. The most common reasons for secondary amputation were local recurrence (LR) and surgical site infection (SSI), with odds ratios of 3.6 and 1.7, respectively. Dogs that underwent secondary amputation had a significantly (P =.05) longer median disease specific survival time (ST) (604 days) compared with the control group (385 days). Dogs lived for a median of 205 days beyond secondary amputation and 97% had good functional outcome. Significant independent factors that positively influenced ST were secondary amputation, moderate SSI, severe SSI and age. [ABSTRACT FROM AUTHOR]