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.

Microscopic characteristics of peri- and postmortem fracture surfaces

AUTHORS

Jessica Skinner, Natalie Langley, Samuel Fahrenholtz, Yuktha Shanavas, Brian Waletzki, Robert Brown, James Herrick, Loukham Shyamsunder, Peter Goguen, Subramaniam Rajan

ABSTRACT

This study investigated if microscopic surface features captured with a scanning electron microscope (SEM) effectively discriminate fracture timing. We hypothesized that microscopic fracture characteristics, including delamination, osteon pullout, and microcracks, may vary as bone elasticity decreases, elucidating perimortem and postmortem events more reliably than macroscopic analyses. Thirty-seven unembalmed, defleshed human femoral shafts from males (n=18) and females (n=2) aged 33–81 years were fractured at experimentally simulated postmortem intervals (PMIs) ranging from 1 to 60 warm weather days (250–40,600 ADH). A gravity convection oven was used to approximate tissue decomposition at 37 C and 27 C, and the resulting heat-time unit (accumulated degree hours, or ADH) was used to examine fractures in elastic/wet versus brittle/dry bone. The bones were fractured with a drop test frame using a three-point bending setup, sensors were used to calculate fracture energy, and high-speed photography documented fracture events. The following data were collected to relate fracture appearance to the biomechanical properties of bone: PMI (postmortem interval) length in ADH, temperature, humidity, collagen percentage, water loss, bone mineral density, cortical bone thickness, fracture energy, age, sex, cause of death, and microscopic fracture feature scores. SEM micrographs were collected from the primary tension zones of each fracture surface, and three microscopic fracture characteristics were scored from a region of interest in the center of the tension zone: percentage of delaminated osteons, percent osteon pullout, and number of microcracks. Multiple linear regression showed that microscopic fracture surface features are strong predictors of ADH (adjusted R-squared=0.67 for the 0 – 40,000 ADH samples; adjusted R-squared=0.92 for the 0–16,000 ADH samples). Osteon pullout is the single best predictor of ADH. Additionally, water loss is the primary driver of bone elasticity changes in low ADH samples, while collagen fibers appear to remain intact until later in the postmortem interval (approximately 40,000 ADH in this study). The results of this study indicate microscopic fracture surface analysis detects the biomechanical effects of decreased elasticity more reliably and with greater sensitivity than macroscopic analysis.

Prkd1 regulates the formation and repair of plasma membrane disruptions (PMD) in osteocytes

AUTHORS

Anik Tuladhar, Joseph C. Shaver, Wesley A. McGee, Kanglun Yu, Jennifer Dorn, J. Luke Horne, Dima W. Alhamad, Mackenzie L. Hagan, Marion A. Cooley, Roger Zhong, Wendy Bollag, Maribeth Johnson, Mark W. Hamrick, Meghan E. McGee-Lawrence

ABSTRACT

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm2), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured.

Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading.

Bone canonical Wnt signaling is downregulated in type 2 diabetes and associates with higher advanced glycation end-products (AGEs) content and reduced bone strength

AUTHORS

Giulia Leanza, Francesca Cannata, Malak Faraj, Claudio Pedone, Viola Viola, Flavia Tramontana, Niccolò Pellegrini, Gianluca Vadalà, Alessandra Piccoli, Rocky Strollo, Francesca Zalfa, Alec T Beeve, Erica L Scheller, Simon Y Tang, Roberto Civitelli, Mauro Maccarrone, Rocco Papalia, Nicola Napoli

ABSTRACT

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156–0.366]) vs non-diabetic subjects 0.352% [0.269–0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46–30.10] vs non-diabetic subjects 76.24 MPa [26.81–132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=–0.7500, p=0.0255; r=–0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s modulus was negatively correlated with SOST (r=−0.5675, p=0.0011), AXIN2 (r=−0.5523, p=0.0042), and SFRP5 (r=−0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.