Mesenchymal Stem Cells-Derived Extracellular Vesicles Mimetics as Osteoinductive Mediators for Bone Healing

AUTHORS

Antoine Karoichan, Ling Li, Celine J. Agnes, Bettina M. Willie, Maryam Tabrizian

ABSTRACT

Bone defects remain challenging to treat, with common therapies still relying on invasive approaches. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) offer a promising alternative due to their regenerative and immunomodulatory properties, but challenges in scalable EV production limit clinical translation. Nanoghosts (NGs) are an emerging class of EV-mimetics synthesized through the physical distortion of ghost cells that offer innate bioactivity similar to EVs while having more scalable yields. In this study, the osteogenic potential of NGs made from MSC ghosts (MSC-NGs) is explored for the first time and contrasted with conventional MSC-EVs. MSC-NGs are generated through sonication, yielding two-fold more vesicles compared to MSC-EVs from the same number of cells. Unlike MSC-EVs, MSC-NGs significantly enhanced the osteogenic differentiation of MSCs, evidenced by increased alkaline phosphatase (ALP) activity and early mineralization. Proteomic analysis further revealed that MSC-NGs are more enriched in osteogenesis-related proteins than MSC-EVs. In vivo, treatment of a 0.5 mm mouse femoral osteotomy with MSC-NGs accelerated fracture healing, showing increased callus mineralization by day 14 and improved bone marrow reconstitution by day 21, along with reduced osteoclastic activity. These findings demonstrate MSC-NGs as scalable and effective therapeutics for bone tissue engineering, offering advantages over MSC-EVs in future bone healing strategies.

Aflatoxin B1 contamination reduces the saponins content and anti-osteoporosis efficacy of the traditional medicine Radix Dipsaci

AUTHORS

Shuqin Lu, Qingsong Yuan, Lulu Wang, Dapeng Su, Min Hu, Lanping Guo, Chuanzhi Kang, Tao Zhou, Jinqiang Zhang

ABSTRACT

Ethnopharmacological relevance

The Radix Dipsaci, a traditional Chinese medicine with a history spanning over 2000 years in China, is widely recognized for its hepatorenal tonic properties, musculoskeletal fortifying effects, fracture healing capabilities, and its frequent application in the treatment of osteoporosis. Like many traditional Chinese herbal medicines, preparations from Radix Dipsaci are at risk of contamination by harmful mycotoxins such as aflatoxin B1.

Aims of the study

This study aims to evaluate the impact of aflatoxin B1 contamination on Radix Dipsaci in terms of changes in quality, efficacy of anti-osteoporosis and hepatorenal toxicity.

Materials and methods

The contamination rates and levels of major mycotoxins were determined in 45 batches of Radix Dipsaci samples using UPLC-MS/MS analysis. The total saponin content and the levels of akebia saponin D in Radix Dipsaci and its decoctions were evaluated through high-performance liquid chromatography (HPLC) analysis. Differences in secondary metabolites between samples without any mycotoxin contamination (N-RD) and those contaminated solely by aflatoxin B1 (AFB1-RD) were compared using metabolomics sequencing and analysis. The anti-osteoporotic efficacy of Radix Dipsaci contaminated with aflatoxin B1 was assessed in a murine model of retinoic acid-induced osteoporosis by quantifying bone mineral content and bone mineral density using dual-energy X-ray absorptiometry. Additionally, the hepatorenal toxicity of Radix Dipsaci contaminated with aflatoxin B1 was evaluated using hematoxylin-eosin staining and enzyme-linked immunosorbent assay (ELISA).

Results

The results indicated that aflatoxin B1 (AFB1) was the most frequently detected mycotoxin, found in 37.7% of the Radix Dipsaci samples. AFB1 contamination significantly altered the secondary metabolites of Radix Dipsaci. Specifically, there was a notable decrease in the levels of total saponins and akebia saponin D in the AFB1-contaminated samples, which exhibited a negative correlation with the levels of AFB1 contamination. However, the administration of a water decoction from AFB1-contaminated Radix Dipsaci did not result in significant improvements in bone mineral density, bone mineral salt content, the trabecular number, trabecular area, proportion of trabecular bone volume/tissue volume and trabecular separation in an osteoporosis mouse model. Additionally, we observed that approximately 16.04% of AFB1 could migrate from the raw herbs into the decoction, leading to hepatocyte and kidney cell damage, as well as increased levels of the oxidative stress molecule malondialdehyde and pro-inflammatory cytokines in the liver and kidney tissues of the osteoporosis model mice.

Conclusion

In summary, Radix Dipsaci is highly susceptible to mycotoxin contamination, particularly aflatoxin B1. The contamination of Radix Dipsaci with AFB1 not only impacts their saponin content and anti-osteoporosis effect but also induces hepatotoxicity and nephrotoxicity.

miR4352b a cross-species modulator of SOSTDC1, targets dual pathway to regulate bone health and fracture healing

AUTHORS

Divya Rai, Anirban Sardar, Anuj Raj, Bhaskar Maji, Shikha Verma, Ashish Kumar Tripathi, Sanchita Gupta, Ashish Sharma, Yogeshwar Vikram Dhar, Ritu Trivedi

ABSTRACT

Mutations in SOST can lead to various monogenic bone diseases. Its paralog, SOSTDC1, shares 55 % protein sequence homology and belongs to the BMP antagonist class. Sostdc1−/− mice exhibit distinct effects on cortical and trabecular bone. Genetic polymorphisms in SOSTDC1 impacting peak bone mass makes SOSTDC1 gene, a candidate for influencing BMD variation in humans. SOSTDC1 is upregulated in bone loss conditions, altering BMP-responsive genes and signaling modulators, suggesting its dual BMP/Wnt antagonist role may enhance both pathways. Overexpression of SOSTDC1 confirmed its role as an osteogenic antagonist. Glycine max (Soy)-derived miR4352b, identified for cross-kingdom applications, precisely targets SOSTDC1, a key regulator of bone. SOSTDC1 competitively binds to BMP2 receptor, BMPR1A. Gma-miR4352b suppresses SOSTDC1 expression, enhancing osteogenesis and countering SOSTDC1's inhibition of osteogenic potential. Modeling estrogen deficiency to mimic elevated SOSTDC1 levels, we observed an inverse correlation with SOSTDC1 expression, while serum BMP2 and PINP levels increased following gma-miR4352b supplementation. In fracture healing, SOSTDC1's crucial role becomes evident in conditions of delayed fracture healing. As healing progresses, SOSTDC1 expression decreases. Gma-miR4352b, compared to scrambled miRNA, remarkably promotes callus formation, achieving 68 % healing by day 10, surpassing the scrambled group at 44 %. By the day 13, the treatment group exhibits advanced healing, challenging to find the callus, while the scrambled group maintains a healing rate similar to day10. The accelerated healing in the treatment group underscores the importance of SOSTDC1 in influencing early fracture healing, potentially through the activation of both BMP2 and Wnt signaling pathways.

Development of miR-26a-activated scaffold to promote healing of critical-sized bone defects through angiogenic and osteogenic mechanisms

AUTHORS

Author links open overlay panelJoanna M. Sadowska a 1, Monika Ziminska b 1, Cole Ferreira c 1, Austyn Matheson a, Auden Balouch c, Jasmine Bogle c, Samantha Wojda c, John Redmond d, Ahmed Elkashif b, Nicholas Dunne b d e f g, Helen O. McCarthy b, Seth Donahue c, Fergal J. O'Brien

ABSTRACT

Very large bone defects significantly diminish the vascular, blood, and nutrient supply to the injured site, reducing the bone's ability to self-regenerate and complicating treatment. Delivering nanomedicines from biomaterial scaffolds that induce host cells to produce bone-healing proteins is emerging as an appealing solution for treating these challenging defects. In this context, microRNA-26a mimics (miR-26a) are particularly interesting as they target the two most relevant processes in bone regeneration-angiogenesis and osteogenesis. However, the main limitation of microRNAs is their poor stability and issues with cytosolic delivery. Thus, utilising a collagen-nanohydroxyapatite (coll-nHA) scaffold in combination with cell-penetrating peptide (RALA) nanoparticles, we aimed to develop an effective system to deliver miR-26a nanoparticles to regenerate bone defects in vivo. The microRNA-26a complexed RALA nanoparticles, which showed the highest transfection efficiency, were incorporated into collagen-nanohydroxyapatite scaffolds and in vitro assessment demonstrated the miR-26a-activated scaffolds effectively transfected human mesenchymal stem cells (hMSCs) resulting in enhanced production of vascular endothelial growth factor, increased alkaline phosphatase activity, and greater mineralisation. After implantation in critical-sized rat calvarial defects, micro CT and histomorphological analysis revealed that the miR-26a-activated scaffolds improved bone repair in vivo, producing new bone of superior quality, which was highly mineralised and vascularised compared to a miR-free scaffold. This innovative combination of osteogenic collagen-nanohydroxyapatite scaffolds with multifunctional microRNA-26a complexed nanoparticles provides an effective carrier delivering nanoparticles locally with high efficacy and minimal off-target effects and demonstrates the potential of targeting osteogenic-angiogenic coupling using scaffold-based nanomedicine delivery as a new "off-the-shelf" product capable of healing complex bone injuries.

Do bone elasticity and postmortem interval affect forensic fractographic analyses?

AUTHORS

Jessica Skinner, Natalie Langley, Malin Joseph, James Herrick, Robert Brown, Brian Waletzki, Peter Goguen Dipl, Loukham Shyamsunder, Subramaniam Rajan

ABSTRACT

Forensic fractographic features of bone reliably establish crack propagation in perimortem injuries. We investigated if similar fracture surface features characterize postmortem fractures. Experimentally induced peri- and postmortem fractures were used to assess if fractographic features vary as bone elasticity decreases during the postmortem interval (PMI). Thirty-seven unembalmed, defleshed human femoral shafts from males and females aged 33–81 years were fractured at varying PMIs with a drop test frame using a three-point bending setup and recorded with a high-speed camera. Vital statistics, cause of death, PMI length, temperature, humidity, collagen percentage, water loss, fracture energy, and fractography scores were recorded for each sample. Results showed that fractographic features associated with perimortem fractures were expressed in PMIs up to 40,600 accumulated degree hours (ADH), or 60 warm weather days. Hackle was the most consistently expressed feature, occurring in all fractures regardless of ADH. The most variable characteristics were wake features (78.4%) and arrest ridges (70.3%). Collagen percentage did not correlate strongly with ADH (r = −0.04, p = 0.81); however, there was a strong significant correlation between ADH and water loss (r = 0.74, p < 0.001). Multinomial logistic regression showed no association between fractographic feature expression and ADH or collagen percentage. In conclusion, forensic fractographic features reliably determine initiation and directionality of crack propagation in experimentally induced PMIs up to 40,600 ADH, demonstrating the utility of this method into the recent postmortem interval. This expression of reliable fractographic features throughout the early PMI intimates these characteristics may not be useful standalone features for discerning peri- versus postmortem fractures.

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

AUTHORS

Pei Hu, Jennifer A. McKenzie, Evan G. Buettmann, Nicole Migotsky, Michael J. Gardner, Matthew J.Silva

ABSTRACT

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.