The treatment of ONFH remains a formidable challenge for orthopedic surgeons, prompting ongoing efforts to identify optimal therapies. In the context of preventing corticosteroid-associated ONFH, certain agents, such as anticoagulants, lipid-lowering agents, and antioxidants, have demonstrated preventive effects in animal studies. However, clinical trials are lacking, representing a significant hurdle in this area of research [25].
Reports have indicated that ONFH can manifest within 1 weeks following corticosteroid administration, highlighting the critical window for preventive measures immediately preceding corticosteroid use [26] [27]. In our study, we administered exosomes concurrently with the induction of the ONFH model to evaluate their potential preventive effects.
ADSC-Exos are emerging as promising candidates due to their ability to facilitate angiogenesis and osteogenesis via paracrine mechanisms. ADSCs, sourced from adipose tissue, possess attributes, such as high proliferative capacity, widespread availability, and multipotency [28, 29]. However, direct application of ADSCs is hampered by challenges, including lengthy cell culture requirements, concerns over ectopic differentiation, and potential immune responses [30].
Exosomes derived from ADSCs offer a viable alternative therapy by harnessing their paracrine-mediated effects. They demonstrate therapeutic efficacy comparable to ADSCs without the associated risks [11]. Moreover, exosome therapy presents several advantages over cell-based treatments. Exosomes exhibit lower immunogenicity and toxicity, making them safer and more feasible for long-term use [31]. They also offer easier storage and greater cost-effectiveness compared to cell-based therapies [32].
The main methods of exosome extraction include ultracentrifugation, size-based techniques, and bead-based immunoprecipitation [33]. Ultrafiltration is a method for purifying exosomes from culture supernatants using only filtration techniques, without ultracentrifugation or special agents. This process takes less time than ultracentrifugation and does not require special equipment, potentially increasing work efficiency and yielding a high recovery rate of exosomes [34, 35]. In this study, we confirmed that highly concentration of exosomes can be extracted from the culture supernatants of two types of hADSC by filter ultrafiltration. Therefore, ultrafiltration is a useful method in exosome retrieval.
In this study, hADSC-Exos demonstrated early reduction of empty lacunae and prevented the progression of ONFH. Moghassemi et al. reported that corticosteroid-induced osteonecrosis is primarily attributed to thrombus-associated ischemia caused by femoral head congestion and induction of apoptosis due to oxidative stress [36]. In vitro studies have demonstrated that ADSC-Exos mitigate osteocyte apoptosis and prevent osteoclast activity by decreasing the expression of receptor activator of nuclear factor kappa b ligand [37]. Additionally, extracellular vesicles from mesenchymal stem cell-conditioned medium act as senomorphics, downregulating senescence-related genes, such as p16INK4a, p21, and p53 [38]. Therefore, hADSC-Exos likely inhibit ONFH progression through protective and anti-apoptotic effects on osteocytes immediately upon administration.
In this study, the exosome-treated group tended to exhibit lower stages in the Modified Ficat and Arlet classification. However, the small sample size (n = 6) used for evaluating empty lacunae and bone mass may limit the ability to detect significant differences in disease stage. Future studies with larger sample sizes could provide more conclusive evidence regarding the efficacy of exosomes in reducing disease severity.
Angiogenesis is critical for prevention of osteonecrosis and bone regeneration. Increased angiogenesis has shown a therapeutic effect of ONFH in steroid-induced ONFH models [39] [40]. ADSC-Exos reportedly promotes angiogenesis in many tissues by enhancing secretion of vascular endothelial growth factor from vascular endothelial cells [41, 42]. Exosomes reportedly inhibit thrombus formation by promoting plasminogen activator inhibitor 1 expression via microRNAs [43]. These effects of thrombus inhibition, and promotion of angiogenesis in ischemic areas may prevent progression of ONFH. Therefore, we expected that angiogenesis would be promoted in the exosome group. Vascular reduction was suppressed in the exosome group, but there was variability in the amount of blood vessels and no significant promotion was observed in this study. This may be attributed to the limited angiogenic effect of exosomes in hypoxic conditions.
In this study, inhibition of secondary OA changes by hADSC-Exos was observed. Recent reports have suggested that hADSC-Exos can downregulate inflammation and oxidative stress, and protect chondrocytes from apoptosis [44] [45] [46]. This may influence the chondrocyte metabolism associated with joint destruction and inhibit the progression of cartilage destruction.
In the early stage of ONFH, the trabecular thickness tends to decrease, but no significant change of the cancellous structure was observed using µCT evaluation within 42 weeks after glucocorticoid administration [2]. This timeframe of 12 weeks may have been insufficient to detect structural changes. Moreover, the measurement of trabecular bone structure was potentially influenced by the localized effects of CD, which could have affected the measurement results.
Wang et al. reported that the administration of exosomes alone show no significant effect likely due to their rapid excretion [47]. Addressing this challenge, researchers have explored methods to establish sustained delivery systems for exosomes [48]. For instance, it has been demonstrated that incorporating FGF-2 into gelatin hydrogel enables long-term sustained release of FGF-2, thereby contributing to the improvement of ONFH [49, 50]. Several studies have reported that encapsulating exosomes in hydrogels can maintain local concentration for long periods of time, thus providing therapeutic benefits [47, 51, 52]. In this study, exosomes were encapsulated in a gelatin gel and placed locally. However, the release kinetics of exosomes from the gelatin hydrogel were not investigated. Further research is needed to assess the efficacy of gel-based delivery systems for exosomes in treating ONFH.
This study had several limitations. First, the mechanism behind the effects of exosomes were not examined. In recent studies, the effects and mechanisms of various microRNAs in exosomes were studied. Optimization of miRNAs in the exosome membrane may be a clue to exosome-based therapies. Second, two types of hADSC-Exos were used, and the dosage of each exosome was different. There is a lack of discussion regarding the optimal dosage and delivery system of exosomes, which remains a topic for future research. Third, the small sample size at each endpoint in our study may have limited our ability to detect significant differences in the parameters examined. Fourth, we only evaluated short-term outcomes up to 12 weeks postoperatively. Future studies should include longer follow-up periods to assess the long-term efficacy and durability of the interventions.