Organoid and organoid extracellular vesicles for osteoporotic fractures therapy: Current status and future perspectives

Osteoporosis is a systemic and degenerative disease characterized by low bone mass and fragile microarchitecture, which predispose patients to fragility fractures, also known as osteoporotic fractures (OPF). OPF have become a major social problem that threaten the health of the elderly. Over the past decade, organoids and organoids extracellular vesicles (OEVs) play a significant role in OPF repair. Organoids have been widely used for fractures treatment. Moreover, EVs are promising nanocarriers due to their cell‐free system, stable drug loading capacity, nanometer size, and good biocompatibility. Importanly, compared with traditional EVs, OEVs have more quantity, better physiological effects, and better therapeutic effects. Therefore, the development of organoid and OEVs in the treatment of OPF is of great significance. Here, we summarize the current status and future perspectives of organoids and OEVs, which will provide innovative solutions to OPF repair.


| OPF
Osteoporosis (OP) is a systemic disease characterized by low bone density and fragile bone microarchitecture. 1 Osteoporotic fractures (OPF) has become a major social problem that threatens the health of the elderly population and brings a heavy burden to social and economic development. In addition, patients with OPF are often at risk of delayed fracture union or nonunion. 2 At present, OPF repair is still an urgent problem to be solved in basic and clinical research. For patients with fragility fractures, the current clinical treatment strategy is long-term antiosteoporotic drugs and artificial bone implants. However, long-term administration of anti-osteoporotic drugs can lead to additional complications, such as osteonecrosis of the jaw and atypical femur fractures. 3 The artificial bone implants are limited by the donor and strong immune rejection, and the artificial bone made of biomaterials has the disadvantage of mismatch in stiffness. All of these make there is no good treatment strategy for OPF at this stage, and for this reason, finding a safe and effective biomaterial to be used in the treatment of patients with OPF is an urgent need at this time.

| ORGANOIDS
Organoids, formed by self-induced in vitro 3D culture, are clusters of pluripotent stem cells or organ-specific adult stem cells with specific structures and functions. 4 At the same time, the organoid retains the properties of stem cells and has a broad therapeutic potential. Recently, organoid treatment of bone defects has been successfully applied in animal models. Endochondral ossification is an important stage of bone regeneration. Mesenchymal stem cells (MSCs) are abundantly enriched in bone regeneration sites and continuously differentiate to produce callus. Callus undergoes hypertrophy, calcification, apoptosis, osteogenic progenitor cell recruitment and osteogenic differentiation to achieve new bone production. 5 Based on this stage of bone regeneration, Ouyang et al. 6 used digital light processing printing technology to load hydrogel microspheres with bone marrow MSCs. They then used chondrogenic induction medium to induce MSCs to differentiate and successfully constructed callus organoids. More importantly, efficient and rapid in situ bone regeneration was achieved using callus organoids within 4 weeks.
More and more bone repair composite materials, such as hydrogels, 7 targeted bone biomaterials, 8 nanoengineered biofilms, 9 silk fibroin-based biomaterials, 10 are becoming an important direction of bone repair. Liu et al. 11 designed BMP-2-initiated in vivo osteo-organoid using active biomaterials. The organoid is capable of forming bone marrow-like structures rich in hematopoietic stem and progenitor cells, MSCs, and a variety of immune cells. This organoid largely restored the developmental pattern of bone marrow tissue and was subsequently validated in animal models. These results suggest that cells generated from bone organoids can treat CCl 4induced liver fibrosis and X-ray radiation-induced hematopoietic injury and have great medical potential. In genaral, bone organoids derived from stem cells provide a new idea for the treatment of complex bone diseases, such as OPF.

| EVs
Extracellular vesicles (EVs) are nanocarriers with phospholipid bilayer structures secreted by most cells.
According to the sizes of EVs, they are mainly divided into exosomes (40-160 nm), microvesicles (200-1000 nm), and apoptotic vesicles (500-2000 nm). 12 EVs are a new drug delivery platform with excellent therapeutic and drug delivery potential. 13 The biogenesis and structure of EVs are shown in Figure 1.
In mammals, EVs are formed and released into the extracellular compartment by secondary endocytosis of the cytoplasmic membrane. Firstly, the cytoplasmic membrane undergoes the first endocytic filling to form early sorting endosome. The early-sorting endosome matures into late-sorting endosome through material exchange with endoplasmic reticulum and Golgi apparatus, and the late endosome produces multi-vesicular bodies (MVBs). Subsequently, MVBs bud inward to form intracavitary vesicles (ILVs). Finally, the MVB may bind to the lysosome and degrade or fuse with the plasma membrane to release ILVs into the extracellular compartment, at which the released ILVs become EVs. 14 Initially, EVs were generally considered to be transport carriers for cellular metabolic waste. As the contents of EVs were studied, it was found that EVs are rich in proteins, nucleic acids, and metabolites. 15 In an in vitro study, it was found that miRNAs from EVs changed significantly during bone marrow stromal cells (BMSCs) osteogenic differentiation. 16 Let-7a, miR-199b, miR-148a, miR-203, and miR-229-5p had significant increased, whereas miR-221, miR-155, and miR-885-5p had significant decreased. The EVs produced by osteogenic differentiation of BMSCs play an important regulatory role in the osteogenic differentiation through their specific miRNAs. 17 However, natural EVs have low miRNA content and can play a limited regulatory role. 18 Engineering EVs can improve their therapeutic efficiency. For example, Liu et al. 19 fused miR-26a-5p (a miRNA with osteogenic properties) into EC-Exo through co-culture. The engineering EC-Exo miR−26a−5p was then encapsulated in hyaluronic acid hydrogel together with APY29 (an anti-inflammatory factor) and injected into the fracture site for fracture repair. 20 Moreover, we had also engineered EVs from vascular endothelial cells and EVs from mouse fibroblasts to deliver miR-155 and miR-188 antagonists to improve OP, respectively. 21 Overall, EVs are excellent nanocarriers for the treatment of bonerelated diseases.

| OEVs
In traditional 2D culture, the cells contact can only be done on the same plane. However, organoids are selforganizing 3D cell clusters in vitro. Therefore, the contact of cells is more similar to that of in vivo. Importantly, organoid-derived EVs share 96% RNA similarity to serum-derived EVs. 22 In subsequent cultivation studies, it was also found that the number of EVs collected in 3D cultivation was 1.5-4.5 times higher than that of 2D cultivation. 23 In addition, EVs collected from 2D and 3D incubations were injected into traumatized mice to observe their recovery, respectively. It was found that mice injected with EVs from 3D sources had better recovery in terms of angiogenesis and neurological recovery. 24 Therefore, organoids extracellular vesicles (OEVs) have better physiological and therapeutic effects than that of traditional EVs.
OEVs have been shown to have great therapeutic effects. For example, hESC was used to grow retinal organoids where Müller glial cells were enriched and collected. When Müller glial cells collected from retinal organoids were injected into mice with retinal degenerative diseases, a more significant recovery of visual function was observed. In subsequent studies, it was found that Müller glial cells could exert some neuroprotective function by releasing certain neurotrophic factors. However, the effect of these released neurotrophic factors was transient. It was possible that the transplanted Müller glial cells exerted a greater neuroprotective effect by releasing EVs to control neuronal apoptosis. 25 In conclusion, not only conventional EVs but also organoids and OEVs can be used as therapeutic tools for bone diseases (Figure 2).

| CONCLUSION AND OUTLOOK
The development of bone organoids is still in its initial stage, and the construction of single-functional bone organoids is not the goal of organoid development. The construction of bone organoids that integrate multiple functions, such as bone formation, bone resorption, and hematopoiesis is the ultimate goal of bone organoid development. 26 The microenvironment in organoids is complex and dynamic. Therefore, the construction of integrated functional bone organoids is essential to better mimic in vitro conditions. The solution of 3D vascularization of bone organoids is commonly combined with organoid-chips, which can not only maintain the normal metabolic activities required for 3D tissue culture but also help to achieve the mimicry of specific microphysiological functions of different organs.
Recently, the applications of OEVs for OPF healing are in its infancy. We are currently concentrating on developing bone organoids. When bone organoids are successfully constructed, we can directly extract OEVs with better physiological effects. Moreover, we can also modify OEVs to obtain stronger functions. Although there is still a long way to go before clinical application, the development of OEVs is getting hotter. The extraction schemes of bone OEVs still need to be improved. The mechanisms of bone OEVs in regulating OPF healing, including the dose, frequency, technology, and safety deserve to be studied. In general, bone organoids and their OEVs will be powerful weapons in the treatment of OPF. We highlight the concept that not only bone organoids can be used to treat OPF but also bone OEVs can be applied to treat OPF (Figure 3).

ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (82230071, 82202344); Shanghai Committee of Science and Technology Laboratory Animal Research Project (23141900600); General F I G U R E 3 Schematic illustration of bone organoids extracellular vesicles (OEVs) for the treatment of osteoporotic fractures (OPF). Healthy bone stem cells are extracted to construct boen organoid, and then OEVs are isolated to treat OPF. Figure was created with https:// app.biorender.com/.