Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury.

Promoting bone healing including fracture non-unions are promising targets for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of exogenous vs. endogenous stem cells and their overall contribution to in vivo fracture repair is not well understood. The purpose of this study was to determine the interaction between exogenous and endogenous stem cells during bone healing. This study was conducted using a standardized burr-hole bone injury model in a mesenchymal progenitor cell (MPC) lineage-tracing mouse under normal homeostatic and osteoporotic conditions. Burr-hole injuries were treated with a collagen-I biomaterial loaded with and without labelled induced pluripotent stem cells (iPSCs). Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone healing were examined. It was observed that treatment with iPSCs resulted in muted healing compared to untreated controls in intact mice post-injury. When the cell populations were examined histologically, iPSC-treated burr-hole defects presented with a dramatic reduction in endogenous MPCs and cell proliferation throughout the injury site. However, when the ovaries were removed and an osteoporotic-like phenotype induced in the mice, iPSCs treatment resulted in increased bone formation relative to untreated controls. In the absence of iPSCs, endogenous MPCs demonstrated robust proliferative and osteogenic capacity to undertake repair and this behaviour was disrupted in the presence of iPSCs which instead took on an osteoblast fate but with little proliferation. This study clearly demonstrates that exogenously delivered cell populations can impact the normal function of endogenous stem/progenitor populations during the normal healing cascade. These interactions need to be better understood to inform cell and biomaterial therapies to treat fractures.

Repurposing the Ordering of Routine Laboratory Tests in Hospitalised Medical Patients (RePORT): results of a cluster randomised stepped-wedge quality improvement study.

BACKGROUND: Low-value use of laboratory tests is a global challenge. Our objective was to evaluate an intervention bundle to reduce repetitive use of routine laboratory testing in hospitalised patients. METHODS: We used a stepped-wedge design to implement an intervention bundle across eight medical units. Our intervention included educational tools and social comparison reports followed by peer-facilitated report discussion sessions. The study spanned October 2020-June 2021, divided into control, feasibility testing, intervention and a follow-up period. The primary outcomes were the number and costs of routine laboratory tests ordered per patient-day. We used generalised linear mixed models, and analyses were by intention to treat. RESULTS: We included a total of 125 854 patient-days. Patient groups were similar in age, sex, Charlson Comorbidity Index and length of stay during the control, intervention and follow-up periods. From the control to the follow-up period, there was a 14% (incidence rate ratio (IRR)=0.86, 95% CI 0.79 to 0.92) overall reduction in ordering of routine tests with the intervention, along with a 14% (ß coefficient=-0.14, 95% CI -0.07 to -0.21) reduction in costs of routine testing. This amounted to a total cost savings of $C1.15 per patient-day. There was also a 15% (IRR=0.85, 95% CI 0.79, 0.92) reduction in ordering of all common tests with the intervention and a 20% (IRR=1.20, 95% CI 1.10 to 1.30) increase in routine test-free patient-days. No worsening was noted in patient safety endpoints with the intervention. CONCLUSIONS: A multifaceted intervention bundle using education and facilitated multilevel social comparison was associated with a safe and effective reduction in use of routine daily laboratory testing in hospitals. Further research is needed to understand how system-level interventions may increase this effect and which intervention elements are necessary to sustain results.

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation.

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (109 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.

Absence of p21(WAF1/CIP1/SDI1) protects against osteopenia and minimizes bone loss after ovariectomy in a mouse model.

p21(WAF1/CIP1/SDI1) is a critical sentinel of the cell cycle that plays an important role in determining cell fate with respect to proliferation, differentiation and apoptosis. Recent studies have demonstrated that inhibition/loss of p21 promotes osteo-chondro differentiation in progenitor/stem cells, and that p21 knockout (p21-/-) mice demonstrate enhanced bone regeneration compared to wild-type controls after a non-critical size defect. It was therefore hypothesized that the absence of p21 may also protect against bone loss through enhancing bone formation, tilting the balance away from bone resorption, in an ovariectomy-induced osteopenia mouse model, investigated via microCT imaging. While p21-/- mice demonstrated significantly less bone loss after ovariectomy compared to wild-type controls, no increase in the number osteoclasts or osteoblasts in the bone or bone marrow was observed, nor was there an increase in osteoclast activity. Therefore, while the absence of p21 protected mice against estrogen mediated bone loss, the mechanisms/pathways responsible remained elusive. This study demonstrates that p21 may play a significant role in bone remodeling, and a better understanding of how the p21 pathway regulates bone anabolism and catabolism could lead to novel therapies for osteoporosis in the future.