A novel biologically hierarchical hydrogel with osteoblast precursor‐targeting extracellular vesicles ameliorates bone loss in vivo via the sequential action of antagomiR‐200b‐3p and antagomiR‐130b‐3p

Abstract Osteoporotic fracture is a major health problem plaguing the ageing society, and improving its treatment is an urgent challenge. How to ameliorate bone loss determines the recovery of such fractures. Extracellular vesicle (EV)‐loaded hydrogel has the capacity to treat osteoporotic fractures due to its pro‐osteogenic property. And balancing proliferation and maturation of osteoblast precursors (OBPs) is of great significance to avoid OBP depletion, which is lacking in current treatment. Based on osteoblastogenic miRNAs, this study aimed to explore the efficacies of the combination of hierarchical hydrogel and EVs altering functional miRNAs level in bone loss. Through bioinformatics analyses, we screened out proliferative gene‐targeting miR‐200b‐3p and osteogenic gene‐targeting miR‐130b‐3p. And antagomiR‐200b‐3p (ant‐200b) enhanced OBP proliferation, and antagomiR‐130b‐3p (ant‐130b) promoted OBP differentiation. After confirming the directional effect of Fibronectin (Fn1) on OBPs, we prepared OBP‐targeting EVs. Furthermore, encapsulation of two antagomiRNAs in EVs enhanced the respective effect of ant‐200b and ant‐130b. Notably, hierarchically injectable hydrogel exerted an effective function in promoting the sequential delivery of EVs‐200b and EVs‐130b. Importantly, hierarchical hydrogel containing dual EVs effectively ameliorated bone loss. Overall, hierarchical hydrogel based on two antagomiRNAs effectively improves bone loss in vivo due to its role in promoting OBP proliferation and maturation sequentially.

the capacity to treat osteoporotic fractures due to its pro-osteogenic property. And balancing proliferation and maturation of osteoblast precursors (OBPs) is of great significance to avoid OBP depletion, which is lacking in current treatment. Based on osteoblastogenic miRNAs, this study aimed to explore the efficacies of the combination of hierarchical hydrogel and EVs altering functional miRNAs level in bone loss. Notably, hierarchically injectable hydrogel exerted an effective function in promoting the sequential delivery of EVs-200b and EVs-130b. Importantly, hierarchical hydrogel containing dual EVs effectively ameliorated bone loss. Overall, hierarchical hydrogel based on two antagomiRNAs effectively improves bone loss in vivo due to its role in promoting OBP proliferation and maturation sequentially.
Hanhao Dai, Yunlong Yu and Junyong Han should be considered joint first authors.

| INTRODUCTION
As a systemic skeletal disorder, osteoporosis is characterized by progressive bone destruction and low bone mass. 1 Osteoporotic fracture is a high-risk factor causing disability and death, which has resulted in serious social problems. 1 Osteoporosis-related fractures, including hip and spinal fractures, have a significant impact on patients' daily activities. 2 Moreover, patients with hip fractures have a two-fold increased risk of mortality. 3 And vertebral fractures also pose a serious threat to the survival of patients. 4 Notably, the mortality rate of patients with secondary fractures has increased by approximately 80%-90%. 3 According to statistics, the fracture caused by osteoporosis is as high as 30% in the women and 11% in the men ≥50 years, which has buried a major hidden danger for the aging society. 3 Bone health depends on the dynamic balance between bone formation dominated by osteoblasts and bone absorption dominated by osteoclasts. 1 The destruction of the above balance can lead to the degradation of bone fibres and increased bone fragility, eventually leading to osteoporosis and subsequent fractures. 1 Therefore, the prevention and treatment of bone loss is the key to improve osteoporotic fracture. Previous studies have demonstrated that the increase of bone mineral density (BMD) in the hip, femoral neck and vertebra can effectively prevent hip and vertebral fractures, and reduce the risk of disability and death in patients with osteoporosis. [5][6][7][8] Accordingly, in the treatment of osteoporotic fractures, more attention should be paid to low bone mass. Biomaterials-related therapeutic strategies that can increase BMD locally are urgently required.
Numerous studies have shown that extracellular vesicles (EVs)loaded hydrogels can locally improve osteogenesis, thereby enhancing bone reconstruction. Wu et al. encapsulated bone mesenchymal stem cells (BMSC)-derived EVs into the chitosan/β-glycerophosphate hydrogel to repair rat calvarial defects. 9 In addition, several studies reported that MSC-derived EVs are loaded into different injectable hydrogels, resulting in improving angiogenesis and osteogenesis, which was used to promote bone regeneration in rat bone defect models. [10][11][12][13] To our knowledge, few studies reported the application of EV-loaded injectable hydrogels in the improvement of hip or vertebral BMD. Therefore, based on EVloaded hydrogels, it is of great scientific significance to seek a local improvement scheme for BMD to prevent hip and vertebral fractures.
Modifying EVs by altering the cargos in EVs is an effective approach to enhance the therapeutic effects of EVs. Hydrogel-based nanobiomaterials can be used to load and deliver osteoporosis drug parathyroid hormone (PTH), which was reported in several studies regarding bone regeneration. 14-16 PTH has significant efficacy in treating osteoporosis due to its pro-osteogenic property. Thus, PTH has been widely used in clinical treatment. However, some disadvantages exist in PTH treatment. Long-term application of PTH may cause several side effects, including nausea, leg cramps, and osteosarcoma. [17][18][19] Also, withdrawal of PTH can lead to abnormal enhancement of adipogenic differentiation of BMSCs. 20 Most importantly, PTH can only promote the osteogenic differentiation while having no effects on the proliferation of osteoblast precursors (OBPs), [21][22][23] thus resulting in the exhaustion of OBPs, which becomes a serious disadvantage in PTH treatment. Following the maturation from OBPs to osteoblasts (OBs), how to retain enough OBPs has become a significant challenge. Accordingly, the therapeutic strategy on balancing the proliferation and maturation of OBPs has become a more perfect scheme for the treatment of osteoporosis.
Some microRNAs (miRNAs) are known to have the capacity to regulate the osteogenesis and proliferation of OBPs. Several miRNAs can suppress the differentiation of OBPs to mature OBs, all of which are attributed to the combination of the 3 0 -untranslated region (3 0 -UTR) of runt-related transcription factor 2 (RUNX2). [24][25][26] Furthermore, the synthesis of miR-188 inhibitor into EVs can effectively improve the repressed osteogenesis caused by corresponding miRNA. 27 In addition, some other miRNAs are responsible for the reduction in the proliferation of OBPs by targeting corresponding proliferation-related genes. [28][29][30][31][32] Accordingly, screening novel miRNAs that could regulate the proliferation and osteogenic differentiation of OBPs by using bioinformatics methods is essential for balancing the proliferation and maturation of OBPs during the treatment of osteoporosis.
Sex-determining region Y-box 2 (Sox2) and Runx2 are key genes regulating the proliferation and osteogenic differentiation of OBPs, 33,34 respectively. In this study, we identified that Sox2-targeting miRNA,

| MATERIALS AND METHODS
A detailed description of all materials and methods can be found in Supporting Information S1: materials and methods.

| Prediction of differentially expressed miRNAs targeting proliferative and osteogenic genes
First, we needed to screen out specific miRNAs having the capacity to regulate the proliferation and osteogenic differentiation of OBPs. Due to the lack of OBP-related databases, we conducted relevant F I G U R E 1 Legend on next page. bioinformatics analyses on homogenetic OBP-derived cells and performed in vitro functional verification. The differentially expressed miRNAs in young and aged BMSCs were identified through bioinformatics analysis. It was found that compared with young BMSCs, 159 miRNAs were upregulated, whereas 184 miRNAs were downregulated in aged BMSCs in GSE57127 with a jlog 2 FCj cutoff criteria >0.5 and p value < 0.05 ( Figure 1A,B). Next, the proliferative genetargeting miRNAs ( Figure 1C) and osteogenic gene-targeting miRNAs ( Figure 1D) were predicted using miRWalk 3.0, TargetScan and miRDB database, and the gene-miRNA pairs were visualized using chord diagrams. Then, the abnormally upregulated miRNAs in aged BMSCs were overlapped with the proliferative gene-targeting miR-NAs ( Figure 1E) and the osteogenic gene-targeting miRNAs ( Figure 1F). Subsequently, qRT-PCR was performed to evaluate the levels of top 3 upregulated proliferative gene-targeting miRNAs and osteogenic gene-targeting miRNAs in P1 and P5 OBPs. Detection of proliferative gene-targeting miRNAs showed that the levels of miR-129-5p (about 2.4 times; Figure 1G), miR-200b-3p (about 3.7 times; Figure 1H) and miR-1896 (about 2.3 times; Figure 1I) in P5 OBPs were greater than those in P1 OBPs. And the upregulation of osteogenic gene-targeting miRNAs was also confirmed: miR-130b-3p (about 10.4 times; Figure 1J), miR-17-3p (about 2.1 times; Figure 1K) and miR-148a-3p (about 1.7 times; Figure 1L). As the two most upregulated miRNAs, miR-200b-3p and miR-130b-3p were selected for further investigation. As shown in the dual-luciferase assays, miR- To verify the reliability of this experimental system, it was necessary to evaluate the proliferative and osteogenic abilities of P5 OBPs.
β-gal senescence-related staining showed that there existed more β-gal positive cells in P5 OBPs than in P1 OBPs ( Figure S1A,B), which confirmed the senescence of P5 OBPs. And the protein level of p16, a marker of irreversible senescence, was markedly increased in P5 OBPs, while the protein expression of Sox2 was significantly decreased in P5 OBPs ( Figure S1C). We found that the proliferative potential was decreased in P5 OBPs presented as less cell viability in CCK8 assay

| Encapsulation of antagomiRNAs in OBPtargeting EVs enhanced the respective effect of ant-200b and ant-130b
The protein detection of recognized EV markers, NTA assays and TEM was performed to identify BMSC-derived EVs. The EVs highly expressed the EV markers, CD9, CD81 and TSG 101, and weakly expressed the negative control marker Calnexin ( Figure 4A). NTA assays showed that the EVs were mainly distributed in 50-120 nm ( Figure 4B), and it was observed that under TEM, the EVs presented a spherical shape ( Figure 4C). Importantly, the results of biotin pulldown assays and Shotgun proteomics showed that Fn1 on the surface of

| Hierarchically injectable hydrogel sequentially delivered EVs-200b and EVs-130b in vivo
The respective role of EVs-200b or EVs-130b in OBP proliferation or osteogenesis was known. Accordingly, we experimentally prepared a new biological hydrogel, which leads to the in vivo controlled release of EVs-200b and EVs-130b sequentially by hierarchical injection. The designed hydrogel contains SA in the inner layer and PF-127 in the outer layer. The characteristics of SA and PF-127 at different concentrations were examined. We found that 3% SA gelled immediately after preparation, while 1% and 2% SA took about 12 and 7 s to form a gel, respectively ( Figure 7A,B). The pore diameter of hydrogel decreased with increased concentration (Figure 7A), and the release sustainability of DiD-labelled EVs mixed in 3% SA was obviously superior to those of the other two concentrations of SA ( Figure 7E,F).
Moreover, the gel-time of 30% PF-127 is significantly faster compared to 20% and 25% PF-127 ( Figure 7C,D). Furthermore, 30% PF-127 had a more compact microstructure ( Figure 7C). Notably, 30% PF-127 showed excellent performance in the sustained release of     loss. In addition, it is worth noting that the current treatment schemes have defects in maintaining the balance between OBP proliferation and osteogenesis, leading to low efficiency in treating bone loss. [21][22][23] Therefore, it is urgent to develop a new type of hydrogel based on OBP proliferation and osteogenic differentiation in an orderly manner.

| Hierarchical hydrogel with EVs-200b and EVs-130b attenuated bone loss in vivo
Our study developed a revolutionary biological hydrogel, causing the sequential release of the above functional miRNA inhibitors, which solves the previous problems related to osteogenesis and effectively alleviates bone loss in mice in vivo.
In this study, we mined two functional miRNAs through bioinfor-  [40][41][42] Previous studies showed that EVs can attach to related cells through the overexpression of specific molecules, which interact with corresponding membrane proteins. [43][44][45][46] This study clari- outer layer, respectively. 48 The above design can sequentially regulate the immunomodulatory microenvironment and bone regeneration to ameliorate bone-implant osseointegration. 48 Similar results were also presented in other studies. [49][50][51] In addition, the injectable hydrogel could prevent EVs from rapid clearance and avoid the multiple injections of EVs in vivo. 52 Here, EVs-200b and EVs-130b were loaded into the hierarchically injectable hydrogel to extend the half-life of the EVs, thereby enhancing the therapeutic effects of corresponding EVs on improving bone mass in vivo. This novel biomaterial is expected to be suitable for the local increase of BMD to prevent osteoporotic fractures in corresponding sites.
There are still some limitations in our study. First, the hierarchically injectable hydrogel was prepared with SA and PF-127, which were moulded after injection. Therefore, the shape and thickness of these two hydrogels are difficult to accurately control. The above problems can be solved through the research and development of the injectable reagent based on hierarchical nanoparticles due to its promotion of the precise release of drugs. [53][54][55] Furthermore, the time to observe the effects of the EV-loaded hydrogel on the bone mass in the OVX mice was 1 month after hydrogel injection, which was much shorter than that of hydrogel degradation in vivo. In the future, how to prolong the action time of this biological hydrogel is an urgent problem to be solved.

| CONCLUSION
In the present study, we constructed OBP-targeting EVs by overex- All authors read and approved the final manuscript.