Promoting Radial Repair in Rabbits through BMP-9 Overexpression in Adipose-Derived Stem Cells Loaded with ICA/nHAC/PLA

Promoting Radial Repair in Rabbits through BMP-9 Overexpression in Adipose-Derived Stem Cells Loaded with ICA/nHAC/PLA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Promoting Radial Repair in Rabbits through BMP-9 Overexpression in Adipose-Derived Stem Cells Loaded with ICA/nHAC/PLA Fang-Tian Xu, Wen-xian Lin, Shuang-Yi Li, Zhong-Hong Lai, Hui-Xian Wei, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5437494/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Bone defects commonly cause delayed bone healing and local dysfunction. Both adipose-derived stem cells (ADSCs) and icariin (ICA)-loaded nano-hydroxyapatite/collagen/polylactic acid (nHAC/PLA) have been reported to promote osteoblast metabolism and accelerate fracture healing. Aim The aim of this study was to explore the mechanism of ADSCs combined with ICA-loaded nHAC/PLA in regulating bone defects. Methods The effects of ICA and BMP-9 (Bone morphogenetic protein-9) overexpression on cell viability and osteogenic differentiation of ADSCs were measured by qRT-PCR, western blotting and alkaline phosphatase (ALP) assay. Alizarin red staining examined the formation of mineralized nodules in ADSCs. Finally, radial defect of rabbit model was established, and administrated by ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA. The new bone formation was detected by X-ray, immunohistochemistry and Hematoxylin and eosin staining. Results BMP-9 overexpression significantly increased ALP activity and mineralized nodule formation in ADSCs, effects that were amplified by ICA. This combination also upregulated the expression of osteogenic markers (BMP-9, OCN, OPN, COL-1, BSP, Runx-2) and influenced key signaling pathways (p38 MAPK, JNK, ERK). In vivo, the treatment group showed enhanced radial bone repair. Conclusions Our findings indicate that the combination of BMP-9-overexpressed ADSCs and ICA-loaded nHAC/PLA scaffolds is effective in promoting bone repair, offering a potential therapeutic strategy for bone defects. Core tip BMP-9 overexpression promoted osteogenic differentiation and proliferation of ADSCs. BMP-9 overexpression combined with ICA promoted osteogenesis in ADSCs. ADSCs with BMP-9 overexpression and ICA-loaded nHAC/PLA promoted radial repair. BMP9 Adipose-derived stem cells ICA nHAC/PLA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The bone loss caused by trauma, congenital malformation or bone tumor surgery is called bone defect. Due to the existence of bone defect, bone nonunion usually leads to delayed union or local dysfunction, which brings great challenges to clinicians [ 1 ] . The main treatment for bone defect is bone grafting. Autogenous cancellous bone grafts are commonly used to treat limited bone defects. However, the critical condition for autogenous repair is exceeded when the defect length reaches 1.5 times the diameter of the diaphysis [ 2 , 3 ] . Traditional treatment of large bone defects includes vascularized fibula transplantation and open bone transplantation. These techniques have a long treatment cycle and are prone to complications such as fracture nonunion. Thus, it is urgent to find a new and effective treatment for bone defect. Adipose-derived stem cells (ADSCs) are a kind of stem cells isolated from adipose tissues with the potential of self-renewal, proliferation and multidirectional differentiation, which can effectively improve tissue defects by promoting cell regeneration [ 4 – 7 ] . Our team's previous research found that ADSCs can not only differentiate into osteoblasts and chondrocyte [ 8 , 9 ] ,but possibly secrete a variety of pro-angiogenic and anti-apoptotic factors to resist oxidative stress damage [ 10 ] . It is expected to be used to repair damaged tissues. Studies have found that ADSCs can proliferate stable in vitro and have a low decay rate. At the same time, ADSCs can be obtained from a small amount of tissue and suitable for large-scale culture [ 11 ] . Thus, ADSCs have gradually become one of the new research focuses in recent years. Additionally, bone morphogenetic protein 9 (BMP-9) has strong osteogenic ability, and plays an important role in inducing osteogenic differentiation of ADSCs [ 12 , 13 ] . Thus, we speculated that BMP-9 may promote the osteogenic differentiation of ADSCs for the treatment of bone defects. Icariin (ICA), a flavonoid glycoside, is the main active ingredient of Epimedium. Modern pharmacological studies have revealed that ICA has a wide range of physiological activities and pharmacological effects, such as neurodegenerative disease, cardiovascular disease, anti-osteoporosis, anti-inflammation, anti-oxidative stress, anti-depression and anti-tumor [ 14 ] . Previous animal studies have shown that ICA promotes the proliferation and differentiation of osteoblasts [ 15 ] .Yan et al reported that Adipose-derived stem cell exosomes loaded with ICA alleviates rheumatoid arthritis by modulating macrophage polarization in rats and attenuated M1 polarization of macrophages via inhibiting the TLR4/Myd88/NF-κB signaling pathway [ 16 , 17 ] .Meanwhile, ICA inhibits the differentiation and maturation of osteoclasts to maintain bone regeneration and indirectly promote fracture healing. With the development of tissue engineering technology, bioactive materials have been the focus of bone defect repair. As a three-dimensional porous biomimetic scaffold material, nano-hydroxyapatite/collagen/polylactic cacid (nHAC/PLA) has a structure similar to artificial bone [ 18 , 19 ] . nHAC/PLA scaffold has good biocompatibility with human body, and participates in the metabolism of osteoblasts in the fracture part through the scaffold bone conduction. Thus, nHAC/PLA may promote callus formation and accelerate fracture healing [ 14 , 20 ] . Previous studies confirmed the potential therapeutic effects of BMP9, ICA and nHAC/PLA in long bone diaphysis defects [ 21 – 23 ] . However, BMP-9-overexpressed ADSCs combined with ICA-loaded nHAC/PLA in the treatment of bone defect have not been reported [ 24 , 25 ] . Therefore, this work constructed a rabbit model of radial bone defect, and attempted to observe the function of BMP-9-overexpressed ADSCs combined with ICA-loaded nHAC/PLA in bone repair, which may provide a new therapy for the repair of bone defect. Animals and methods Animals New Zealand white rabbits (eight weeks old, weighting 1.8 ± 0.3 Kg) were obtained from Hua Fukang Biological Technology (Beijing, China). Rabbits were housed under SPF conditions and allowed to take food and water freely. All procedures involving animals were approved by The Medical Ethics Committee of Gannan Medical University (2015048). Isolation and culture of ADSCs Rabbits were anesthetized with 15% chloral hydrate. Under a sterile environment, the subcutaneous adipose tissue separated from the groin of rabbits. The visible blood vessels and fascia were removed from the adipose tissues. After washing with PBS, the subcutaneous adipose tissue was cut into small pieces and digested by 0.25% type I collagenase (Sigma, St. Louis, USA) for 30 min. The cell suspension was cultured in DMEM (Beyotime, Shanghai, China) containing 10% foetal bovine serum (FBS; CLARK Bioscience, Australia) and 1% penicillin-streptomycin (Sigma, St. Louis, USA). When the cells grow to 90% fusion rate, the cells were digested with 0.25% trypsin (Beiotim, Shanghai, China) and inoculated in fresh medium for further culture. The medium was replaced every three days. Identification of ADSCs ADSCs were collected and washed with 1 x Perm/Wash buffer. The cells were blocked with 5% goat serum (Beyotime) at 4℃ for 15 min. Cells were incubated with anti-CD29, anti-CD31, anti-CD44 (Cell Signaling Technology, Boston, USA) at 4℃ in dark for 30 min. After washing with 1 x Perm/Wash buffer for several times, cells were resuscitated the staining buffer. The expression of CD29, CD31 and anti-CD44 was detected by flow cytometry. Cell treatment ADSCs at logarithmic phase were treated with different concentrations (0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/ml) of ICA (Yuanye Biotechnology, Shanghai, China) at 37℃ and 5% CO 2 for 24 h. CCK-8 assay ADSCs at logarithmic phase were inoculated in a 96-well plate and treated with different concentrations of ICA at 37℃ in 5% CO 2 . After 24 hours of incubation, 20 μL CCK8 assay (Beyotime) was added to each well and cultured at 37℃ for 1 h. The OD value of each well was measured at 450 nm wavelength by a microplate analyzer. Cell infection The pcDNA3.1 vector carrying BMP-9 was packaged into lentivirus (LV) particles (LV-BMP-9). ADSCs were seeded into a 96-well plate and cultured 37℃ and CO 2 for overnight. The cells were infected with LV-BMP-9 (GenePharma, Shanghai, China) for 48 h. After 6 hours of incubation, the medium was replaced with DMEM high glucose containing 10% FBS and 1% penicillin-streptomycin. Finally, the cells were observed under a microscope and photographed. Alkaline phosphatase（ALP）activity determination ADSCs were inoculated into 96-well plates and cultured at 37℃ and 5% CO 2 for 48h. Then, ADSCs were treated with different concentrations (6.25, 12.5 and 25 µg/ml) of ICA for 24 h. The cells supernatant was collected and the ALP (Nanjing Jiancheng Bioengineering Institute, China) activity was detected based on the kit instructions. Alizarin red staining First, the 24 well of osteoblast induced cell plates were covered by 4% gelatin for 1 h at room temperature. After the gelatin was removed, the ADSCs were inoculated in 24-well plates and cultured at 37℃ and 5% CO 2 for 24 h. After the cells wash with PBS twice, each well was fixed with 400 μL 70% ethanol at room temperature for 1h. Then, 300 μL 1% alizarin red dye (Yuanye Biotechnology, Shanghai, China) was added to each well and incubated at room temperature for 30 min. The cells were observed under an optical microscope. RNA extraction and real-time PCR ADSCs were lysed with Trizol lysate (Beyotime) in the centrifuge tube without RNA enzyme at room temperature for 5 min. After centrifugation at 12000 rpm for 15 min, the upper water phase was transferred to a centrifuge tube without RNA enzyme. Isopropyl alcohol of equal volume was added and the RNA precipitates were observed after being placed in the refrigerator at -20℃ for 30 min. RNA samples were served as template to synthesize complementary DNA utilizing PrimeScript RT reagent kit (TaKaRa, Shiga, Japan). PCR reaction was carried out applying TB Green® Premix Ex Taq™ (TaKaRa). Amplification conditions: 94℃ for 10 min, 94℃ for 20 s 55℃ for 20 s, 72℃ for 20 s. Data were analyzed using the 2 -ΔΔCt method. The primer sequence (5’-3’) was shown as follows: BMP-9: Forward-GAGGCAGTTEAGGACCTCAG and Reverse-CTTAGGCAGGAGACGGTCAG; OCN: Forward-GACACCATGAGGACCCTCTC and Reverse-GCCTGGTAGTTGTTGTGAGC；OPN：Forward-GCCGTGATTTGCTTTTGTC and Reverse-TTCATTGGAGTCCTGGTTA；COL-I：Forward-GCTTCTCATTCTCATGGATG and Reverse-GCAGCAATGACAACAAGAC; Runx-2：Forward-GCTTGATGACTCTAAACCT and Reverse-AATCTGACTCTGTCCTTGT; GAPDH：Forward-ACCTGACCTGCCGCCTGGAGAAAGC and Reverse-GGAGACGACCTGGTCCTCGGTGTAG. Rabbit model of radial defect Twelve New Zealand white rabbits were randomly divided into 6 groups (n = 2). The periosteum was cut 10 mm from the middle part of the radius to expose the interception site and the bone defect model was established by the dental drill. The surgical site was disinfected by injection of penicillin daily for three days. Radial defect rabbits were divided into 6 groups: radial defect model, radial defect rabbits received nHAC/PLA treatment, radial defect rabbits received nHAC/PLA +ADSCs treatment, radial defect rabbits received nHAC/PLA +ADSCs+ICA treatment, radial defect rabbits received nHAC/PLA +ADSCs+BMP-9 (25 μg/ml) treatment and radial defect rabbits received nHAC/PLA +ADSCs+BMP-9(25 μg/ml) +ICA treatment. The nHAC/PLA +ADSCs+BMP-9(25 μg/ml) +ICA group was photographed by X-ray at 0 d, 30 d, 60 d and 90 d after surgery and the other five groups were photographed by X-ray at 90 d after surgery. New Zealand white rabbits were sacrificed at 90 d after operation and normal bone tissues of at least 0.5 cm around the bone defect area were taken for further experiments. Protein extraction and Western blotting (WB) ADSCs and bone tissues were lysed with RIPA (Beyotime) for 2 h. Each sample was added 200 μL BCA working solution (Beyotime). The protein concentration was measured under microplate reader at 562 nm. The protein samples were separated by 8-12% SDS-PAGE gel electrophoresis, and then transferred onto the PVDF membranes (Millipore, Billerica, USA). The membranes were soaked in methanol for 1 min and sealed with 5% skimmed milk containing 0.1% Tween-20 (Solarbio, Beijing, China) at room temperature for 1 h. The membranes were incubated with primary antibody at 4℃ overnight and then cultured with secondary antibody at room temperature for 1 h. Finally, the chemiluminescence of membranes was developed by ECL reagent (Beyotime). The protein antibodies used in this study were listed as follows: anti-BMP-9 (1:300, Cell Signaling Technology), anti-BSP (1:500, Cell Signaling Technology), anti-OCN (1:300), anti-OPN (1:500, Santa Cruz, CA, USA), anti-Runx-2 (1:1000, Santa Cruz), anti-ERK1/2 (1:1000, Santa Cruz), anti-P38 (1:1000, Cell Signaling Technology), anti-JNK (1:2000, Santa Cruz), anti-p-ERK1/2 (1:2000, Proteintech, Wuhan, China), anti-p-p38 (1:2000, Proteintech), anti-p-JNK (1:2000, Proteintech), anti-Smad1 (1:1000, Cell Signaling Technology), anti-p-Smad1 (1:2000, Proteintech), anti-GAPDH (1:2000, Proteintech). Immunohistochemical staining Bone tissues were fixed with 4% paraformaldehyde, and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4) for 4 weeks. Then, bone tissues were dehydrated with different concentration of ethanol and embedded in paraffin for further analysis. The paraffin specimens were cut into 5μm thick sections. After dewaxing and dehydration, the paraffin sections were treated with 3% H 2 O 2 at room temperature for 10 min. The paraffin sections were incubated with 0.1% Trion X-100 (Beyotime) at room temperature for 10 min and rinsed with PBS for 5 min. After blocking with 5% bull serum albumin at 37 ℃ for 1 h, the paraffin sections were incubated with anti-VEGF, anti-TGF-β and anti-BMP-9 at 4℃ overnight, and then stained with secondary antibody at room temperature for 1 h. The sections were stained with DAB dye solution (Solarbio). Hematoxylin and eosin (HE) staining After dewaxing and dehydration, the paraffin sections of bone tissues were washed with distilled water and stained with hematoxylin (Solarbio). The dyeing time was generally controlled about 5 min according to the condition of tissue staining. The sections were washed with distilled water until it was blue and purple. The sections were differentiated with 1% hydrochloric acid ethanol for 2 s until the tissue turns red. The sections were stained with eosin for 8 s. After dehydration with anhydrous ethanol, the images were observed and photographed under the microscope. Statistical analysis All data was represented as mean ± standard deviation. The data was analyzed by SPSS 18.0 and the image was processed by Prism 9.0. Two independent sample T test was used to detect statistically significant differences between the two groups and multivariate analysis of variance (ANOVA) was used for comparison of multiple groups. P <0.05 was considered to indicate a statistically significant difference. RESULTS Identification of rabbit ADSCs ADSCs were isolated from New Zealand rabbits. ADSCs were initially small and round. After 24 hours of incubation, cells were almost completely attached to the wall. The third generation of cells were long and narrow, arranged orderly and oriented. As shown in Figure 1A, part of the cells developed spindle-shaped or triangular antennae. Flow cytometry showed that the ADSCs in P3 generation were positive for the mesenchymal markers for CD29 and CD44 and negative for the vascular endothelial cell marker CD31 (Figure 1B). ICA inhibited cell viability of ADSCs CCK-8 assay examined the influence of ICA on cell viability of ADSCs. The results showed that the ICA repressed cell viability of ADSCs in a concentration-dependent manner. The toxicity of ICA at 50 µg/ml to ADSCs was more than 50%. The analysis of cell growth curve showed that the IC50 value of ICA was 33.67 µg/ml (Figure 2A-B). BMP-9 overexpression combined with ICA treatment promoted the formation of mineralized nodules in ADSCs ADSCs were infected with LV-BMP-9 at 10, 50 and 100 of MOI. As shown in Figure 3A, the infection efficiency of LV-BMP-9 increased with the increase of MOI value. The infection efficiency reached about 90% when the MOI value was 100. Excessive LV will have toxic effect on cells. Therefore, LV-BMP-9 at 100 MOI was used to infect ADSCs (Figure 3A). The activity of ALP in ADSCs was detected. BMP-9 overexpression significantly increased the ALP activity of ADSCs. Compared with BMP-9 group, BMP-9 overexpression combined with ICA treatment (6.25, 12.5 and 25 µg/ml) did not further promote ALP activity of ADSCs (Figure 3B). Applying alizarin red staining, it showed that BMP-9 up-regulation promoted the formation of mineralized nodules in ADSCs, which was further accelerated by ICA treatment in a concentration-dependent manner (6.25, 12.5 and 25 µg/ml) (Figure 3C). Thus, ICA at 25 µg/ml was used to treat ADSCs in further assays. BMP-9 overexpression combined with ICA treatment induced osteogenic differentiation and proliferation Results obtained from Alizarin red staining showed that BMP-9 overexpression enhanced the number of mineralized nodules in ADSCs, which was further increased by ICA treatment (25 μg/ml) (Figure 4A). Moreover, qRT-PCR was performed to examine the expression of osteogenesis genes and proteins in ADSCs. Both BMP-9 overexpression and LV-BMP-9 combined with ICA elevated the expression of BMP-9, OCN, OPN, COL-1 and Runx2 in ADSCs. Compared with BMP-9 group, higher expression of BMP-9, OCN, COL-1 and Runx2 was observed in ADSCs in the presence of BMP-9 overexpression and LV-BMP-9 combined with ICA (Figure 4B). WB results indicated that the expression of BMP-9, BSP, OCN, OPN, and Runx-2 in ADSCs was elevated by both BMP-9 overexpression and LV-BMP-9 combined with ICA. Compared with BMP-9 group, LV-BMP-9 combined with ICA caused an increase of BMP-9, BSP, OCN and Runx-2 in ADSCs (Figure 4C). BMP-9 overexpression combined with ICA treatment promoted osteogenesis via activating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways We subsequently demonstrated the specific pathways by which ICA and BMP-9 regulate osteogenesis. Alizarin red staining examined the formation of mineralized nodules in ADSCs. BMP-9 overexpression combined with ICA treatment elevated the formation of mineralized nodules in ADSCs, which was further increased by PD98059 treatment and inhibited by SB203580 and SP600125 treatment (Figure 5A). Moreover, BMP-9 overexpression combined with ICA treatment enhanced the activity of ALP in ADSCs. Compared with BMP-9+ICA group, the activity of ALP in ADSCs was enhanced by PD98059 treatment and inhibited by SB203580 and SP600125 treatment (Figure 5B). The qRT-PCR assessed the expression of OCN, COL-1 and Runx-2 in ADSCs. PD98059 treatment led to an up-regulation of OCN, COL-1 and Runx-2 in ADSCs in the presence of LV-BMP-9+ICA treatment. SB203580 and SP600125 reduced the expression of OCN, COL-1 and Runx-2 in ADSCs following LV-BMP-9+ICA treatment (Figure 5C). WB results showed that the protein expression of p-Smad1, p-p38 and p-JNK was increased in PD98059 + BMP-9+ ICA group, while the protein expression of p-ErK1/2 was decreased. Nevertheless, the protein expression of p-Smad1, p-P38 and p-JNK was decreased in the SB203580 + BMP-9+ ICA group and SP600125 + BMP-9+ ICA group, while the protein expression of p-ERK1/2 was increased (Figure 5D). Thus, BMP-9 overexpression combined with ICA treatment effectively promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways. ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted bone repair in vivo In order to verify the biological role of ADSCs+BMP-9+ICA+nHAC/PLA in bone loss in vivo , we established bone defect model (Supplementary Figure 1). Compared with radial defect model of rabbit, the average gray value of X ray was increased in nHAC/PLA, nHAC/PLA +ADSCs, nHAC/PLA +ADSCs+ICA, nHAC/PLA +ADSCs+BMP-9 and nHAC/PLA +ADSCs+BMP-9+ICA groups, especially nHAC/PLA +ADSCs+BMP-9+ICA group. As time goes on, the gray value of nHAC/PLA +ADSCs+BMP-9+ICA group also continued to increase (Figure 6A). The qRT-PCR results showed that the mRNA expression of BMP-9, OCN, TGF-β and Runx-2 in the nHAC/PLA+ADSCs+BMP-9+ICA group were higher than that in nHAC/PLA group (Figure. 6B). Moreover, WB results showed that the protein expressions of BMP-9, OCN and Runx-2 in the nHAC/PLA+ADSCs+BMP-9+ICA group were higher than nHAC/PLA group (Figure. 6C). Immunohistochemical staining results showed that compared with nHAC/PLA group, nHAC/PLA+ADSCs+BMP-9+ICA increased the expression of BMP-9, OCN, TGF-β in radial defect rabbits (Figure 7A-C). Finally, HE staining examined the pathological changes of bone tissues of rabbits. HE staining showed that nHAC/PLA+ADSCs+BMP-9+ICA further increased the osteocyte content and promoted a large number of bone trabecular formation in bone tissues of radial defect rabbits (Figure 8A-B). DISCUSSION Long bone defect is a clinical problem faced by orthopedic surgeons. This work attempted to explore a new treatment of bone loss. We found that BMP-9 overexpression enhanced the activity of ALP, promoted the formation of mineralized nodules and elevated the expression of BMP-9, OCN, OPN, COL-1, BSP and Runx-2 in ADSCs. These influences conferred by BMP-9 overexpression was further accelerated by ICA treatment. BMP-9 overexpression combined with ICA treatment promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways. In vivo , ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted radial repair. Currently, iliac auto-graft is used for limited bone defects. Previous researches have confirmed that BMP-9 is the most effective bioactive factor with the ability of ectopic osteogenesis [ 19 , 26 ] . BMP-9 is a bioactive functional protein that belonging to the TGF-β family, which regulates the proliferation and differentiation of osteoblasts [ 27 ] . BMP-9 not only induce ADSCs to differentiate into bone-related tissue and cartilage, but also directly affects the expression of ADSCs and the activity of ALP. BMP-9 plays an important role in the process of fracture repair and has been widely used in the repair of bone defect [ 28 , 29 ] . In this work, we found that BMP-9 overexpression the activity of ALP and promoted the osteogenesis of ADSCs. Moreover, BMP-9 overexpression led to an up-regulation of osteogenic genes and proteins, OCN, OPN, COL-1, BSP and Runx-2 in ADSCs. A large number of biological materials have been developed for bone defect treatment [ 30 ] . In recent years, most studies have focused on composite biomaterials, including the effective active ingredient cytokines or traditional Chinese medicine loaded in biomaterials, to better promote the repair of bone defects [ 31 ] . Traditional Chinese medicine ICA is a flavonoid glycoside with the function of strengthening muscle and bone [ 32 ] . Currently, ICA has been proved to promote proliferation of and osteogenic differentiation of bone marrow mesenchymal stem cells and adipose-derived stem cells [ 33 , 34 ] . ICA loading of nHAC/PLA in biomaterials containing growth factors for the treatment of bone loss deficiency is a promising research strategy [ 35 , 36 ] . Consistently, this work also confirmed that ICA treatment elevated the activity of ALP and promoted osteogenic differentiation of ADSCs. ICA treatment also further elevated the promotion of BMP-9 overexpression on the osteogenesis of ADSCs. In vivo , ICA-loaded nHAC/PLA combined with BMP-9-overexpressed ADSCs promoted radial repair in radial defect rabbits. MAPK kinase is a conserved serine/threonine protein kinase in eukaryotic cells. ERK, P38 and JNK are three subfamilies of MAPK, which mainly transduces cell-surface stimulating signals to the nucleus and regulate cell proliferation, differentiation and apoptosis [ 37 , 38 ] . ERK regulates osteoblast proliferation and apoptosis by regulating cell cycle expression and the activity of the osteoblast specific transcription factor Runx2 [ 39 , 40 ] . Meanwhile, inhibition of ERK signaling pathway regulates osteoblast differentiation. Recent studies have shown that activation of P38 MAPK and JNK pathways promotes the expression of ALP and the proliferation of osteoblasts to regulate bone formation [ 41 – 43 ] . This study showed that p38 MAPK and JNK and ERK signaling pathways participated in the osteogenesis of ADSCs. BMP-9 overexpression combined with ICA treatment promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways. CONCLUSION This work demonstrated that ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted radial repair in rabbits. Therefore, this study suggested that BMP-9 overexpressed ADSCs combined with ICA-loaded nHAC/PLA may be a potential treatment for bone defects, which provides a theoretical basis for its application in clinical research. In the future, we will focus on exploring its further clinical application to provide patients with better treatment. Declarations Author contributions: Hui-Xian Wei and Ling-Zhang Meng conceptualized and designed this study. Wen-xian Lin and Shuang-Yi collected and processed the data. Shuang-Yi and Zhong-hong Lai analyzed and interpreted the data. Fang-Tian Xu and Wen-xian Lin wrote the manuscript. Fang-Tian Xu, Wen-Xian Lin, and Shuang-Li contributed equally to this work. All authors reviewed and approved the final version of the manuscript. Funding This work was supported by the Guangxi Natural Science Foundation (2023GXNSFDA026035), the National Nature Science Foundation of China (82260433,81560358) and the Youth Science Innovation and Entrepreneurship Talent Training Project of Nanning (RC20220108). Footnotes Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at Gannan Medical University. Institutional animal care and use committee statement: All procedures involving animals were approved by The Medical Ethics Committee of Gannan Medical University (2015048). Declaration of interests The authors declare that there is no conflict of interests. Data sharing statement The data involved in this study can be obtained from the corresponding author. 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Evaluation of BMP-2 Enhances the Osteoblast Differentiation of Human Amnion Mesenchymal Stem Cells Seeded on Nano-Hydroxyapatite/Collagen/Poly(l-Lactide). Int J Mol Sci. 2018. 19(8): 2171. Tang ZB, Cao JK, Wen N, et al. Posterolateral spinal fusion with nano-hydroxyapatite-collagen/PLA composite and autologous adipose-derived mesenchymal stem cells in a rabbit model. J Tissue Eng Regen Med. 2012. 6(4): 325-36. Liu X, Liu HY, Lian X, et al. Osteogenesis of mineralized collagen bone graft modified by PLA and calcium sulfate hemihydrate: in vivo study. J Biomater Appl. 2013. 28(1): 12-9. Calixto RD, Freitas GP, Souza PG, et al. Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair. J Cell Physiol. 2023. 238(11): 2625-2637. E LL, Zhang R, Li CJ, et al. Effects of rhBMP-2 on Bone Formation Capacity of Rat Dental Stem/Progenitor Cells from Dental Follicle and Alveolar Bone Marrow. Stem Cells Dev. 2021. 30(8): 441-457. Bharadwaz A, Jayasuriya AC. Osteogenic differentiation cues of the bone morphogenetic protein-9 (BMP-9) and its recent advances in bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2021. 120: 111748. Pinto K, Tim CR, Crovace MC, et al. Scaffolds of bioactive glass-ceramic (Biosilicate®) and bone healing: A biological evaluation in an experimental model of tibial bone defect in rats. Biomed Mater Eng. 2018. 29(5): 665-683. Liu L, Chen Y, Song D, Huang D. BMP9 is a potential therapeutic agent for use in oral and maxillofacial bone tissue engineering. Biochem Soc Trans. 2020. 48(3): 1269-1285. Fujioka-Kobayashi M, Kobayashi E, Schaller B, Mottini M, Miron RJ, Saulacic N. Effect of recombinant human bone morphogenic protein 9 (rhBMP9) loaded onto bone grafts versus barrier membranes on new bone formation in a rabbit calvarial defect model. J Biomed Mater Res A. 2017. 105(10): 2655-2661. Wang Y, Wang R, Zhang F. Icariin promotes the proliferation and differentiation of osteoblasts from the rat mandible by the Wnt/β‑catenin signalling pathway. Mol Med Rep. 2018. 18(3): 3445-3450. Wang Y, Zhang H, Qiang H, et al. Innovative Biomaterials for Bone Tumor Treatment and Regeneration: Tackling Postoperative Challenges and Charting the Path Forward. Adv Healthc Mater. 2024. 13(16): e2304060. Guo Y, Chi X, Wang Y, et al. Mitochondria transfer enhances proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cell and promotes bone defect healing. Stem Cell Res Ther. 2020. 11(1): 245. Teng JW, Bian SS, Kong P, Chen YG. Icariin triggers osteogenic differentiation of bone marrow stem cells by up-regulating miR-335-5p. Exp Cell Res. 2022. 414(2): 113085. Ye Y, Jing X, Li N, Wu Y, Li B, Xu T. Icariin promotes proliferation and osteogenic differentiation of rat adipose-derived stem cells by activating the RhoA-TAZ signaling pathway. Biomed Pharmacother. 2017. 88: 384-394. Khosravipour A, Amini A, Masteri Farahani R, et al. Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats. Biochem Biophys Res Commun. 2020. 531(2): 105-111. Yin N, Wang Y, Ding L, et al. Platelet-rich plasma enhances the repair capacity of muscle-derived mesenchymal stem cells to large humeral bone defect in rabbits. Sci Rep. 2020. 10(1): 6771. Tóthová Z, Šemeláková M, Solárová Z, Tomc J, Debeljak N, Solár P. The Role of PI3K/AKT and MAPK Signaling Pathways in Erythropoietin Signalization. Int J Mol Sci. 2021. 22(14): 7682. Park HB, Baek KH. E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers. Biochim Biophys Acta Rev Cancer. 2022. 1877(3): 188736. Liu Q, Zhuang Y, Ouyang N, Yu H. Cytochalasin D Promotes Osteogenic Differentiation of MC3T3-E1 Cells via p38-MAPK Signaling Pathway. Curr Mol Med. 2019. 20(1): 79-88. Wang Y, Shen S, Hu T, et al. Layered Double Hydroxide Modified Bone Cement Promoting Osseointegration via Multiple Osteogenic Signal Pathways. ACS Nano. 2021. 15(6): 9732-9745. Chen J, Yu M, Li X, Sun QF, Yang CZ, Yang PS. Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling. J Periodontal Res. 2020. 55(3): 363-373. Kim W, Tokuda H, Tanabe K, et al. Acetaminophen reduces osteoprotegerin synthesis stimulated by PGE(2) and PGF(2α) in osteoblasts: attenuation of SAPK/JNK but not p38 MAPK or p44/p42 MAPK. Biomed Res. 2021. 42(2): 77-84. Xie B, Zeng Z, Liao S, Zhou C, Wu L, Xu D. Kaempferol Ameliorates the Inhibitory Activity of Dexamethasone in the Osteogenesis of MC3T3-E1 Cells by JNK and p38-MAPK Pathways. Front Pharmacol. 2021. 12: 739326. Additional Declarations No competing interests reported. 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\u0026 The People’s Hospital of Guangxi Zhuang Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Wen-xian","middleName":"","lastName":"Lin","suffix":""},{"id":377808993,"identity":"fcad1603-f840-41c0-8b4f-8cef1d440ba5","order_by":2,"name":"Shuang-Yi Li","email":"","orcid":"","institution":"Liaoning University of Traditional Chinese Medicine Xinglin College","correspondingAuthor":false,"prefix":"","firstName":"Shuang-Yi","middleName":"","lastName":"Li","suffix":""},{"id":377808994,"identity":"561c4b04-3f83-4aa2-8b40-8167b6ef32b7","order_by":3,"name":"Zhong-Hong Lai","email":"","orcid":"","institution":"The First Affiliated Hospital of Gannan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhong-Hong","middleName":"","lastName":"Lai","suffix":""},{"id":377808995,"identity":"1a34b206-0ff3-4213-a93d-8f229edcfcc6","order_by":4,"name":"Hui-Xian Wei","email":"","orcid":"","institution":"The Ninth People's Hospital of 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version.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5437494/v1/6824c397f7b23d78afe198c7.png"},{"id":95529237,"identity":"b9010cf2-f683-40ba-b301-5401aa2743b2","added_by":"auto","created_at":"2025-11-10 10:16:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18289723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5437494/v1/a5efd16a-40ef-45f1-a421-dde9e999188f.pdf"},{"id":71199807,"identity":"afdd0b31-7417-4964-b963-0a9ef1f2363b","added_by":"auto","created_at":"2024-12-12 06:01:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":401566,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5437494/v1/1f4fe6c0256276a2de6a8d5c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Promoting Radial Repair in Rabbits through BMP-9 Overexpression in Adipose-Derived Stem Cells Loaded with ICA/nHAC/PLA","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe bone loss caused by trauma, congenital malformation or bone tumor surgery is called bone defect. Due to the existence of bone defect, bone nonunion usually leads to delayed union or local dysfunction, which brings great challenges to clinicians\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The main treatment for bone defect is bone grafting. Autogenous cancellous bone grafts are commonly used to treat limited bone defects. However, the critical condition for autogenous repair is exceeded when the defect length reaches 1.5 times the diameter of the diaphysis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Traditional treatment of large bone defects includes vascularized fibula transplantation and open bone transplantation. These techniques have a long treatment cycle and are prone to complications such as fracture nonunion. Thus, it is urgent to find a new and effective treatment for bone defect.\u003c/p\u003e \u003cp\u003eAdipose-derived stem cells (ADSCs) are a kind of stem cells isolated from adipose tissues with the potential of self-renewal, proliferation and multidirectional differentiation, which can effectively improve tissue defects by promoting cell regeneration\u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Our team's previous research found that ADSCs can not only differentiate into osteoblasts and chondrocyte\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e,but possibly secrete a variety of pro-angiogenic and anti-apoptotic factors to resist oxidative stress damage\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. It is expected to be used to repair damaged tissues. Studies have found that ADSCs can proliferate stable \u003cem\u003ein vitro\u003c/em\u003e and have a low decay rate. At the same time, ADSCs can be obtained from a small amount of tissue and suitable for large-scale culture\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Thus, ADSCs have gradually become one of the new research focuses in recent years. Additionally, bone morphogenetic protein 9 (BMP-9) has strong osteogenic ability, and plays an important role in inducing osteogenic differentiation of ADSCs\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Thus, we speculated that BMP-9 may promote the osteogenic differentiation of ADSCs for the treatment of bone defects.\u003c/p\u003e \u003cp\u003eIcariin (ICA), a flavonoid glycoside, is the main active ingredient of Epimedium. Modern pharmacological studies have revealed that ICA has a wide range of physiological activities and pharmacological effects, such as neurodegenerative disease, cardiovascular disease, anti-osteoporosis, anti-inflammation, anti-oxidative stress, anti-depression and anti-tumor\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Previous animal studies have shown that ICA promotes the proliferation and differentiation of osteoblasts\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.Yan et al reported that Adipose-derived stem cell exosomes loaded with ICA alleviates rheumatoid arthritis by modulating macrophage polarization in rats and attenuated M1 polarization of macrophages via inhibiting the TLR4/Myd88/NF-κB signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.Meanwhile, ICA inhibits the differentiation and maturation of osteoclasts to maintain bone regeneration and indirectly promote fracture healing.\u003c/p\u003e \u003cp\u003eWith the development of tissue engineering technology, bioactive materials have been the focus of bone defect repair. As a three-dimensional porous biomimetic scaffold material, nano-hydroxyapatite/collagen/polylactic cacid (nHAC/PLA) has a structure similar to artificial bone\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. nHAC/PLA scaffold has good biocompatibility with human body, and participates in the metabolism of osteoblasts in the fracture part through the scaffold bone conduction. Thus, nHAC/PLA may promote callus formation and accelerate fracture healing \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies confirmed the potential therapeutic effects of BMP9, ICA and nHAC/PLA in long bone diaphysis defects\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, BMP-9-overexpressed ADSCs combined with ICA-loaded nHAC/PLA in the treatment of bone defect have not been reported\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, this work constructed a rabbit model of radial bone defect, and attempted to observe the function of BMP-9-overexpressed ADSCs combined with ICA-loaded nHAC/PLA in bone repair, which may provide a new therapy for the repair of bone defect.\u003c/p\u003e"},{"header":"Animals and methods","content":"\u003cp\u003eAnimals\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNew Zealand white rabbits (eight weeks old, weighting 1.8 \u0026plusmn; 0.3 Kg) were obtained from Hua Fukang Biological Technology (Beijing, China). Rabbits were housed under SPF conditions and allowed to take food and water freely. All procedures involving animals were approved by The Medical Ethics Committee of Gannan Medical University (2015048).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIsolation and culture of ADSCs\u003c/p\u003e\n\u003cp\u003eRabbits were anesthetized with 15% chloral hydrate. Under a sterile environment, the subcutaneous adipose tissue separated from the groin of rabbits. The visible blood vessels and fascia were removed from the adipose tissues. After washing with PBS, the subcutaneous adipose tissue was cut into small pieces and digested by 0.25% type I collagenase (Sigma, St. Louis, USA) for 30 min. The cell suspension was cultured in DMEM (Beyotime, Shanghai, China) containing 10% foetal bovine serum (FBS; CLARK Bioscience, Australia) and 1% penicillin-streptomycin (Sigma, St. Louis, USA). When the cells grow to 90% fusion rate, the cells were digested with 0.25% trypsin (Beiotim, Shanghai, China) and inoculated in fresh medium for further culture. The medium was replaced every three days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIdentification of ADSCs\u003c/p\u003e\n\u003cp\u003eADSCs were collected and washed with 1 x Perm/Wash buffer. The cells were blocked with 5% goat serum (Beyotime) at 4℃ for 15 min. Cells were incubated with anti-CD29, anti-CD31, anti-CD44 (Cell Signaling Technology, Boston, USA) at 4℃ in dark for 30 min. After washing with 1 x Perm/Wash buffer for several times, cells were resuscitated the staining buffer. The expression of CD29, CD31 and anti-CD44 was detected by flow cytometry.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell treatment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eADSCs at logarithmic phase were treated with different concentrations (0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 \u0026micro;g/ml)\u0026nbsp;of ICA (Yuanye Biotechnology, Shanghai, China) at 37℃ and 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 24 h.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCCK-8 assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eADSCs at logarithmic phase were inoculated in a 96-well plate and treated with different concentrations of ICA at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 hours of incubation, 20 \u0026mu;L CCK8 assay (Beyotime) was added to each well and cultured at 37℃ for 1 h. The OD value of each well was measured at 450 nm wavelength by a microplate analyzer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell infection\u003c/p\u003e\n\u003cp\u003eThe pcDNA3.1 vector carrying BMP-9 was packaged into lentivirus (LV) particles (LV-BMP-9). ADSCs were seeded into a 96-well plate and cultured 37℃ and CO\u003csub\u003e2\u003c/sub\u003e for overnight. The cells were infected with LV-BMP-9 (GenePharma, Shanghai, China) for 48 h. After 6 hours of incubation, the medium was replaced with DMEM high glucose containing 10% FBS and 1% penicillin-streptomycin. Finally, the cells were observed under a microscope and photographed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlkaline phosphatase（ALP）activity determination\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eADSCs were inoculated into 96-well plates and cultured at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e for 48h. Then, ADSCs were treated with different concentrations (6.25, 12.5 and 25 \u0026micro;g/ml) of ICA for 24 h. The cells supernatant was collected and the ALP (Nanjing Jiancheng Bioengineering Institute, China) activity was detected based on the kit instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlizarin red staining\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirst, the 24 well of osteoblast induced cell plates were covered by 4% gelatin for 1 h at room temperature. After the gelatin was removed, the ADSCs were inoculated in 24-well plates and cultured at 37℃ and 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 24 h. After the cells wash with PBS twice, each well was fixed with 400 \u0026mu;L 70% ethanol at room temperature for 1h. Then, 300 \u0026mu;L 1% alizarin red dye (Yuanye Biotechnology, Shanghai, China) was added to each well and incubated at room temperature for 30 min. The cells were observed under an optical microscope. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRNA extraction and real-time PCR\u003c/p\u003e\n\u003cp\u003eADSCs were lysed with Trizol lysate (Beyotime) in the centrifuge tube without RNA enzyme at room temperature for 5 min. After centrifugation at 12000 rpm for 15 min, the upper water phase was transferred to a centrifuge tube without RNA enzyme. Isopropyl alcohol of equal volume was added and the RNA precipitates were observed after being placed in the refrigerator at -20℃ for 30 min. RNA samples were served as template to synthesize complementary DNA utilizing PrimeScript RT reagent kit (TaKaRa, Shiga, Japan). PCR reaction was carried out applying TB Green\u0026reg; Premix Ex Taq\u0026trade; (TaKaRa). Amplification conditions: 94℃ for 10 min, 94℃ for 20 s 55℃ for 20 s, 72℃ for 20 s. Data were analyzed using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. The primer sequence (5\u0026rsquo;-3\u0026rsquo;) was shown as follows: BMP-9: Forward-GAGGCAGTTEAGGACCTCAG and Reverse-CTTAGGCAGGAGACGGTCAG; OCN: Forward-GACACCATGAGGACCCTCTC and Reverse-GCCTGGTAGTTGTTGTGAGC；OPN：Forward-GCCGTGATTTGCTTTTGTC and Reverse-TTCATTGGAGTCCTGGTTA；COL-I：Forward-GCTTCTCATTCTCATGGATG and Reverse-GCAGCAATGACAACAAGAC; Runx-2：Forward-GCTTGATGACTCTAAACCT and Reverse-AATCTGACTCTGTCCTTGT; GAPDH：Forward-ACCTGACCTGCCGCCTGGAGAAAGC and Reverse-GGAGACGACCTGGTCCTCGGTGTAG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRabbit model of radial defect\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwelve New Zealand white rabbits were randomly divided into 6 groups (n = 2). The periosteum was cut 10 mm from the middle part of the radius to expose the interception site and the bone defect model was established by the dental drill. The surgical site was disinfected by injection of penicillin daily for three days. Radial defect rabbits were divided into 6 groups: radial defect model, radial defect rabbits received nHAC/PLA treatment, radial defect rabbits received nHAC/PLA +ADSCs treatment, radial defect rabbits received nHAC/PLA +ADSCs+ICA treatment, radial defect rabbits received nHAC/PLA +ADSCs+BMP-9 (25 \u0026mu;g/ml) treatment and radial defect rabbits received nHAC/PLA +ADSCs+BMP-9(25 \u0026mu;g/ml) +ICA treatment. The nHAC/PLA +ADSCs+BMP-9(25 \u0026mu;g/ml) +ICA group was photographed by X-ray at 0 d, 30 d, 60 d and 90 d after surgery and the other five groups were photographed by X-ray at 90 d after surgery. New Zealand white rabbits were sacrificed at 90 d after operation and normal bone tissues of at least 0.5 cm around the bone defect area were taken for further experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein extraction and Western blotting (WB)\u003c/p\u003e\n\u003cp\u003eADSCs and bone tissues were lysed with RIPA (Beyotime) for 2 h. Each sample was added 200 \u0026mu;L BCA working solution (Beyotime). The protein concentration was measured under microplate reader at 562 nm. The protein samples were separated by 8-12% SDS-PAGE gel electrophoresis, and then transferred onto the PVDF membranes (Millipore, Billerica, USA). The membranes were soaked in methanol for 1 min and sealed with 5% skimmed milk containing 0.1% Tween-20 (Solarbio, Beijing, China) at room temperature for 1 h. The membranes were incubated with primary antibody at 4℃ overnight and then cultured with secondary antibody at room temperature for 1 h. Finally, the chemiluminescence of membranes was developed by ECL reagent (Beyotime). The protein antibodies used in this study were listed as follows: anti-BMP-9 (1:300, Cell Signaling Technology), anti-BSP (1:500, Cell Signaling Technology), anti-OCN (1:300), anti-OPN (1:500, Santa Cruz, CA, USA), anti-Runx-2 (1:1000, Santa Cruz), anti-ERK1/2 (1:1000, Santa Cruz), anti-P38 (1:1000, Cell Signaling Technology), anti-JNK (1:2000, Santa Cruz), anti-p-ERK1/2 (1:2000, Proteintech, Wuhan, China), anti-p-p38 (1:2000, Proteintech), anti-p-JNK (1:2000, Proteintech), anti-Smad1 (1:1000, Cell Signaling Technology), anti-p-Smad1 (1:2000, Proteintech), anti-GAPDH (1:2000, Proteintech).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining\u003c/p\u003e\n\u003cp\u003eBone tissues were fixed with 4% paraformaldehyde, and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4) for 4 weeks. Then, bone tissues were dehydrated with different concentration of ethanol and embedded in paraffin for further analysis. The paraffin specimens were cut into 5\u0026mu;m thick sections. After dewaxing and dehydration, the paraffin sections were treated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at room temperature for 10 min. The paraffin sections were incubated with 0.1% Trion X-100 (Beyotime) at room temperature for 10 min and rinsed with PBS for 5 min. After blocking with 5% bull serum albumin at 37 ℃ for 1 h, the paraffin sections were incubated with anti-VEGF, anti-TGF-\u0026beta; and anti-BMP-9 at 4℃ overnight, and then stained with secondary antibody at room temperature for 1 h. The sections were stained with DAB dye solution (Solarbio).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHematoxylin and eosin (HE) staining\u003c/p\u003e\n\u003cp\u003eAfter dewaxing and dehydration, the paraffin sections of bone tissues were washed with distilled water and stained with hematoxylin (Solarbio). The dyeing time was generally controlled about 5 min according to the condition of tissue staining. The sections were washed with distilled water until it was blue and purple. The sections were differentiated with 1% hydrochloric acid ethanol for 2 s until the tissue turns red. The sections were stained with eosin for 8 s. After dehydration with anhydrous ethanol, the images were observed and photographed under the microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data was represented as mean \u0026plusmn; standard deviation. The data was analyzed by SPSS 18.0 and the image was processed by Prism 9.0. Two independent sample T test was used to detect statistically significant differences between the two groups and multivariate analysis of variance (ANOVA) was used for comparison of multiple groups. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIdentification of rabbit ADSCs\u003c/p\u003e\n\u003cp\u003eADSCs were isolated from New Zealand rabbits. ADSCs were initially small and round. After 24 hours of incubation, cells were almost completely attached to the wall. The third generation of cells were long and narrow, arranged orderly and oriented. As shown in Figure 1A, part of the cells developed spindle-shaped or triangular antennae. Flow cytometry showed that the ADSCs in P3 generation were positive for the mesenchymal markers for CD29 and CD44 and negative for the vascular endothelial cell marker CD31 (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eICA inhibited cell viability of ADSCs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCCK-8 assay examined the influence of ICA on cell viability of ADSCs. The results showed that the ICA repressed cell viability of ADSCs in a concentration-dependent manner. The toxicity of ICA at 50 \u0026micro;g/ml to ADSCs was more than 50%. The analysis of cell growth curve showed that the IC50 value of ICA was 33.67 \u0026micro;g/ml (Figure 2A-B). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMP-9 overexpression combined with ICA treatment promoted the formation of mineralized nodules in ADSCs \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eADSCs were infected with LV-BMP-9 at 10, 50 and 100 of MOI. As shown in Figure 3A, the infection efficiency of LV-BMP-9 increased with the increase of MOI value. The infection efficiency reached about 90% when the MOI value was 100. Excessive LV will have toxic effect on cells. Therefore, LV-BMP-9 at 100 MOI was used to infect ADSCs (Figure 3A). The activity of ALP in ADSCs was detected. BMP-9 overexpression significantly increased the ALP activity of ADSCs. Compared with BMP-9 group, BMP-9 overexpression combined with ICA treatment (6.25, 12.5 and 25 \u0026micro;g/ml) did not further promote ALP activity of ADSCs (Figure 3B). Applying alizarin red staining, it showed that BMP-9 up-regulation promoted the formation of mineralized nodules in ADSCs, which was further accelerated by ICA treatment in a concentration-dependent manner (6.25, 12.5 and 25 \u0026micro;g/ml) (Figure 3C). Thus, ICA at 25 \u0026micro;g/ml was used to treat ADSCs in further assays. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMP-9 overexpression combined with ICA treatment induced osteogenic differentiation and proliferation\u003c/p\u003e\n\u003cp\u003eResults obtained from Alizarin red staining showed that BMP-9 overexpression enhanced the number of mineralized nodules in ADSCs, which was further increased by ICA treatment (25 \u0026mu;g/ml) (Figure 4A). Moreover, qRT-PCR was performed to examine the expression of osteogenesis genes and proteins in ADSCs. Both BMP-9 overexpression and LV-BMP-9 combined with ICA elevated the expression of BMP-9, OCN, OPN, COL-1 and Runx2 in ADSCs. Compared with BMP-9 group, higher expression of BMP-9, OCN, COL-1 and Runx2 was observed in ADSCs in the presence of BMP-9 overexpression and LV-BMP-9 combined with ICA (Figure 4B). WB results indicated that the expression of BMP-9, BSP, OCN, OPN, and Runx-2 in ADSCs was elevated by both BMP-9 overexpression and LV-BMP-9 combined with ICA. Compared with BMP-9 group, LV-BMP-9 combined with ICA caused an increase of BMP-9, BSP, OCN and Runx-2 in ADSCs (Figure 4C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMP-9 overexpression combined with ICA treatment promoted osteogenesis via activating\u0026nbsp;p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways\u003c/p\u003e\n\u003cp\u003eWe subsequently demonstrated the specific pathways by which ICA and BMP-9 regulate osteogenesis. Alizarin red staining examined the formation of mineralized nodules in ADSCs. BMP-9 overexpression combined with ICA treatment elevated the formation of mineralized nodules in ADSCs, which was further increased by PD98059 treatment and inhibited by SB203580 and SP600125 treatment (Figure 5A). Moreover, BMP-9 overexpression combined with ICA treatment enhanced the activity of ALP in ADSCs. Compared with BMP-9+ICA group, the activity of ALP in ADSCs was enhanced by PD98059 treatment and inhibited by SB203580 and SP600125 treatment (Figure 5B). The qRT-PCR assessed the expression of OCN, COL-1 and Runx-2 in ADSCs. PD98059 treatment led to an up-regulation of OCN, COL-1 and Runx-2 in ADSCs in the presence of LV-BMP-9+ICA treatment. SB203580 and SP600125 reduced the expression of OCN, COL-1 and Runx-2 in ADSCs following LV-BMP-9+ICA treatment (Figure 5C). WB results showed that the protein expression of p-Smad1, p-p38 and p-JNK was increased in PD98059 + BMP-9+ ICA group, while the protein expression of p-ErK1/2 was decreased. Nevertheless, the protein expression of p-Smad1, p-P38 and p-JNK was decreased in the SB203580 + BMP-9+ ICA group and SP600125 + BMP-9+ ICA group, while the protein expression of p-ERK1/2 was increased (Figure 5D). Thus, BMP-9 overexpression combined with ICA treatment effectively promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted bone repair in vivo\u003c/p\u003e\n\u003cp\u003eIn order to verify the biological role of ADSCs+BMP-9+ICA+nHAC/PLA in bone loss \u003cem\u003ein vivo\u003c/em\u003e, we established bone defect model (Supplementary Figure 1). Compared with radial defect model of rabbit, the average gray value of X ray was increased in nHAC/PLA, nHAC/PLA +ADSCs, nHAC/PLA +ADSCs+ICA, nHAC/PLA +ADSCs+BMP-9 and nHAC/PLA +ADSCs+BMP-9+ICA groups, especially nHAC/PLA +ADSCs+BMP-9+ICA group. As time goes on, the gray value of nHAC/PLA +ADSCs+BMP-9+ICA group also continued to increase (Figure 6A). The qRT-PCR results showed that the mRNA expression of BMP-9, OCN, TGF-\u0026beta; and Runx-2 in the nHAC/PLA+ADSCs+BMP-9+ICA group were higher than that in nHAC/PLA group (Figure. 6B). Moreover, WB results showed that the protein expressions of BMP-9, OCN and Runx-2 in the nHAC/PLA+ADSCs+BMP-9+ICA group were higher than nHAC/PLA group (Figure. 6C). Immunohistochemical staining results showed that compared with nHAC/PLA group, nHAC/PLA+ADSCs+BMP-9+ICA increased the expression of BMP-9, OCN, TGF-\u0026beta; in radial defect rabbits (Figure 7A-C). Finally, HE staining examined the pathological changes of bone tissues of rabbits. HE staining showed that nHAC/PLA+ADSCs+BMP-9+ICA further increased the osteocyte content and promoted a large number of bone trabecular formation in bone tissues of radial defect rabbits (Figure 8A-B).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eLong bone defect is a clinical problem faced by orthopedic surgeons. This work attempted to explore a new treatment of bone loss. We found that BMP-9 overexpression enhanced the activity of ALP, promoted the formation of mineralized nodules and elevated the expression of BMP-9, OCN, OPN, COL-1, BSP and Runx-2 in ADSCs. These influences conferred by BMP-9 overexpression was further accelerated by ICA treatment. BMP-9 overexpression combined with ICA treatment promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways. \u003cem\u003eIn vivo\u003c/em\u003e, ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted radial repair.\u003c/p\u003e \u003cp\u003eCurrently, iliac auto-graft is used for limited bone defects. Previous researches have confirmed that BMP-9 is the most effective bioactive factor with the ability of ectopic osteogenesis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. BMP-9 is a bioactive functional protein that belonging to the TGF-β family, which regulates the proliferation and differentiation of osteoblasts\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. BMP-9 not only induce ADSCs to differentiate into bone-related tissue and cartilage, but also directly affects the expression of ADSCs and the activity of ALP. BMP-9 plays an important role in the process of fracture repair and has been widely used in the repair of bone defect\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In this work, we found that BMP-9 overexpression the activity of ALP and promoted the osteogenesis of ADSCs. Moreover, BMP-9 overexpression led to an up-regulation of osteogenic genes and proteins, OCN, OPN, COL-1, BSP and Runx-2 in ADSCs.\u003c/p\u003e \u003cp\u003eA large number of biological materials have been developed for bone defect treatment\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In recent years, most studies have focused on composite biomaterials, including the effective active ingredient cytokines or traditional Chinese medicine loaded in biomaterials, to better promote the repair of bone defects\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Traditional Chinese medicine ICA is a flavonoid glycoside with the function of strengthening muscle and bone\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Currently, ICA has been proved to promote proliferation of and osteogenic differentiation of bone marrow mesenchymal stem cells and adipose-derived stem cells\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. ICA loading of nHAC/PLA in biomaterials containing growth factors for the treatment of bone loss deficiency is a promising research strategy\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Consistently, this work also confirmed that ICA treatment elevated the activity of ALP and promoted osteogenic differentiation of ADSCs. ICA treatment also further elevated the promotion of BMP-9 overexpression on the osteogenesis of ADSCs. \u003cem\u003eIn vivo\u003c/em\u003e, ICA-loaded nHAC/PLA combined with BMP-9-overexpressed ADSCs promoted radial repair in radial defect rabbits.\u003c/p\u003e \u003cp\u003eMAPK kinase is a conserved serine/threonine protein kinase in eukaryotic cells. ERK, P38 and JNK are three subfamilies of MAPK, which mainly transduces cell-surface stimulating signals to the nucleus and regulate cell proliferation, differentiation and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. ERK regulates osteoblast proliferation and apoptosis by regulating cell cycle expression and the activity of the osteoblast specific transcription factor Runx2\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, inhibition of ERK signaling pathway regulates osteoblast differentiation. Recent studies have shown that activation of P38 MAPK and JNK pathways promotes the expression of ALP and the proliferation of osteoblasts to regulate bone formation\u003csup\u003e[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. This study showed that p38 MAPK and JNK and ERK signaling pathways participated in the osteogenesis of ADSCs. BMP-9 overexpression combined with ICA treatment promoted osteogenesis by stimulating p38 MAPK and JNK signaling pathways and inhibiting ERK signaling pathways.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis work demonstrated that ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA promoted radial repair in rabbits. Therefore, this study suggested that BMP-9 overexpressed ADSCs combined with ICA-loaded nHAC/PLA may be a potential treatment for bone defects, which provides a theoretical basis for its application in clinical research. In the future, we will focus on exploring its further clinical application to provide patients with better treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui-Xian Wei and Ling-Zhang Meng conceptualized and designed this study. Wen-xian Lin and Shuang-Yi collected and processed the data. Shuang-Yi and Zhong-hong Lai analyzed and interpreted the data. Fang-Tian Xu and Wen-xian Lin wrote the manuscript. Fang-Tian Xu, Wen-Xian Lin, and Shuang-Li contributed equally to this work. All authors reviewed and approved the final version of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Guangxi Natural Science Foundation (2023GXNSFDA026035), the National Nature Science Foundation of China (82260433,81560358) and the Youth Science Innovation and Entrepreneurship Talent Training Project of Nanning (RC20220108).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitutional review board statement: The study was reviewed and approved by the Institutional Review Board at Gannan Medical University.\u003c/p\u003e\n\u003cp\u003eInstitutional animal care and use committee statement: All procedures involving animals were approved by The Medical Ethics Committee of Gannan Medical University (2015048).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interests. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data involved in this study can be obtained from the corresponding author. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eARRIVE guidelines statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNakamura S, Ito T, Okamoto K, et al. 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Front Pharmacol. 2021. 12: 739326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BMP9, Adipose-derived stem cells, ICA, nHAC/PLA","lastPublishedDoi":"10.21203/rs.3.rs-5437494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5437494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone defects commonly cause delayed bone healing and local dysfunction. Both adipose-derived stem cells (ADSCs) and icariin (ICA)-loaded nano-hydroxyapatite/collagen/polylactic acid (nHAC/PLA) have been reported to promote osteoblast metabolism and accelerate fracture healing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAim\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to explore the mechanism of ADSCs combined with ICA-loaded nHAC/PLA in regulating bone defects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of ICA and BMP-9 (Bone morphogenetic protein-9) overexpression on cell viability and osteogenic differentiation of ADSCs were measured by qRT-PCR, western blotting and alkaline phosphatase (ALP) assay. Alizarin red staining examined the formation of mineralized nodules in ADSCs. Finally, radial defect of rabbit model was established, and administrated by ADSCs with BMP-9 overexpression combined with ICA-loaded nHAC/PLA. The new bone formation was detected by X-ray, immunohistochemistry and Hematoxylin and eosin staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMP-9 overexpression significantly increased ALP activity and mineralized nodule formation in ADSCs, effects that were amplified by ICA. This combination also upregulated the expression of osteogenic markers (BMP-9, OCN, OPN, COL-1, BSP, Runx-2) and influenced key signaling pathways (p38 MAPK, JNK, ERK). In vivo, the treatment group showed enhanced radial bone repair.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings indicate that the combination of BMP-9-overexpressed ADSCs and ICA-loaded nHAC/PLA scaffolds is effective in promoting bone repair, offering a potential therapeutic strategy for bone defects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCore tip\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMP-9 overexpression promoted osteogenic differentiation and proliferation of ADSCs.\u003cstrong\u003e \u003c/strong\u003eBMP-9 overexpression combined with ICA promoted osteogenesis in ADSCs.\u003cstrong\u003e \u003c/strong\u003eADSCs with BMP-9 overexpression and ICA-loaded nHAC/PLA promoted radial repair.\u003c/p\u003e","manuscriptTitle":"Promoting Radial Repair in Rabbits through BMP-9 Overexpression in Adipose-Derived Stem Cells Loaded with ICA/nHAC/PLA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 06:00:17","doi":"10.21203/rs.3.rs-5437494/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"860abbd5-943a-407d-9d31-2ce69f5d7a37","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T07:24:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-12 06:00:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5437494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5437494","identity":"rs-5437494","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}