Autologous ADSCs with exogenous NPY promotes fracture healing in ovariectomized rats

Hong Liu: Writing – review & editing, Writing – original draft, Validation, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jiaoyang Wu: Methodology, Data curation. Huixuan Wu: Methodology, Data curation. Ting Wang: Validation, Software, Resources. Houde Zhou: Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Min Liu: Writing – review & editing, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Conceptualization.

Ethical clearance was obtained from the regional ethical committee of Third Xiangya Hospital, Central South University. The study protocol (including the research question, key design features, and analysis plan) was included in the ethical application. The title of the approved project was “Repair effect of NPY induced adipose derived stem cells on osteoporosis fracture of rats”; and the approval number was 2019-S034 on March 11, 2019. The ethics application and ethics approval documents can be provided if requested.

Not applicable.

This work was supported by the 10.13039/501100004735 Natural Science Foundation of Hunan Province (grant numbers: 2022JJ40749 ); the Project of Scientific Research Plan of Health Committee of Hunan Province (grant numbers: 202203065334 ), the National Natural Scientific Foundation of China [grant numbers: 82170900 ], 10.13039/501100018537 National Science and Technology Major Project  [grant number: 2023ZD0509205 ]； Key Research and Development Program of Hunan [ 2024JK2113 ], Hunan Provincial Health High-Level Talent Scientific Research Project [grant number: R2023019], Hunan Administration of Traditional Chinese Medicine [grant numbers: A2023021 ], and Zhishanyufeng Project of third Xiangya Hospital , 10.13039/501100002822 Central South University .

We successfully established the primary culture model of rats’ ADSCs and amplified it in vitro, providing a cytological research platform for the next step. ADSCs were fusiform and fibroblast-like cells ( Fig. 1 . A) that can be induced to differentiate into osteoblast (Alkaline Phosphatase staining at 14 days and Alizarin Red staining at 21 days) in vitro ( Fig. 1 B and C). Flow cytometry was used to characterize the immunophenotypes of ADSCs (CD44+/CD45-) ( Fig. 1 D). Fig. 1 The primary culture model of rats' ADSCs. A.3rd generation on day 5; B.ALP staining of osteogenic differentiation at 14 days; C.Alizarin red staining of osteogenic differentiation at 21 days; D.flow cytometry identified the surface antigen expression: CD44 + , CD45 − . Fig. 1 The primary culture model of rats' ADSCs. A.3rd generation on day 5; B.ALP staining of osteogenic differentiation at 14 days; C.Alizarin red staining of osteogenic differentiation at 21 days; D.flow cytometry identified the surface antigen expression: CD44 + , CD45 − . We treated ADSCs with NPY (10 −4  M, 10 −6  M, 10 −8  M, 10 −10  M and 10 −12  M) for 48 h, and found that NPY concentrations of 10 −6  M and 10 −4  M significantly inhibited the proliferation of ADSCs ( Fig. S1 ). Finally, we chose the NPY concentrations of 10 −8  M, 10 −10  M and 10 −12  M to treat ADSCs for 14 and 21 days, respectively, to observe osteogenic differentiation. Osteogenic differentiation was significantly promoted by NPY via Alkaline Phosphatase (ALP) staining at 14 days ( Fig. 2 A-C) and Alizarin Red staining at 21 days ( Fig. 2 D-F). NPY also promoted the bone-related genes such as bone morphogenetic protein 2 (BMP2) ( Fig. 2 .G1), osteocalcin (OC) ( Fig. 2 .G2), runt-related transcription factor 2 (runx2) ( Fig. 2 .G3), osterix ( Fig. 2 .G4), and alkaline phosphatase (ALP) ( Fig. 2 .G5). In addition, NPY promoted the protein levels of ALP, runx2, osterix, OC and BMP2 significantly ( Fig. 2 H and Fig. S2 ). There was a dose-dependent promotion effect of NPY on ADSCs’ osteogenic differentiation. Therefore, in the following experiments, we used NPY treated with 10 −8  M. Fig. 2 Efficacy of NPY on osteogenetic differentiation of ADSCs in vitro. A-B, ALP staining at day 14; C, the quantitative result of ALP staining at day 14; D-E, Alizarin red staining at day 21; F, the quantitative result of Alizarin red staining at day 21. A1(B1,C1,D1)-A5(B5,C5,D5): control, classical osteogenic induction medium, classical osteogenic induction medium + NPY10 −8  M(3)/10 −10  M(4)/10 −12  M(5); G–H: osteogenic related mRNA and protein levels, full-length blots were presented in Supplementary Fig. S2 . Fig. 2 Efficacy of NPY on osteogenetic differentiation of ADSCs in vitro. A-B, ALP staining at day 14; C, the quantitative result of ALP staining at day 14; D-E, Alizarin red staining at day 21; F, the quantitative result of Alizarin red staining at day 21. A1(B1,C1,D1)-A5(B5,C5,D5): control, classical osteogenic induction medium, classical osteogenic induction medium + NPY10 −8  M(3)/10 −10  M(4)/10 −12  M(5); G–H: osteogenic related mRNA and protein levels, full-length blots were presented in Supplementary Fig. S2 . The success of OVX in rats was confirmed by the changes of the body weight, uterine size and weight, as well as body BMD. Bilateral ovariectomy ( Fig. 3 A) was conducted. Despite pair-feeding, OVX rats weighed 4%–14 % more than control rats. For example, control and OVX rats weighed 314 ± 9 and 345 ± 7g (p < 0.05), respectively, at 3 months postsurgery (See Supplementary Information ( Fig. S3 ) for quality files). After OVX, the estradiol levels declined and led to decreased uterine size ( Fig. 3 B) and uterine weight by ∼60–72 % after 12 weeks (uterine weight is ∼500–600 mg in rats aged 6 months) ( Fig. 3 C). A slight but significant decrease in BMD was observed in OVX rats compared with sham rats ( Fig. 3 D) at 90 days postsurgery. Fig. 3 The establishment of osteoporotic fracture model. A. OVX, bilateral ovariectomy; B-C. Uterine sizes (B) and uterine weight (C) between OVX and sham groups; D. Total body BMD between OVX and sham groups; E-G. Fracture of tibia in its bottom third. Fig. 3 The establishment of osteoporotic fracture model. A. OVX, bilateral ovariectomy; B-C. Uterine sizes (B) and uterine weight (C) between OVX and sham groups; D. Total body BMD between OVX and sham groups; E-G. Fracture of tibia in its bottom third. We used our own designed equipment, which had been patented, to establish closed fracture of the lower third of the tibia ( Fig. 3 E). The success was confirmed by dissection and observation of the transverse tibial fracture of three randomly chosen rats ( Fig. 3 F). In addition, a postoperative X-ray was used on the following day to confirm the success of the transverse tibial fractures ( Fig. 3 G). Three and six weeks later the fracture healing were assessed by X-ray. X-ray images showed that the fracture line was still clear in the vehicle group at 3-weeks ( Fig. 4 A1-A5 ) post-fracture and was blurred with the extending of time (6-weeks) ( Fig. 4 A6-A10 ). However, the fracture line was blurred and difficult to discern in the NPY-ADSCs group at the same time point ( Fig. 4C1-C5 and Fig. 4C6-C10 ). Fracture healing in the ADSCs group was between the vehicle group and the NPY-ADSCs group ( Fig. 4 B1-B10 ). Fig. 4 Imaging and histological evaluation of the therapeutic effects on 21 and 42 days after treatment. A-C. X-ray and micro-computed tomography (micro-CT); D-F. fast green/saf-O staining for histology 42 days after injection. A1-A14,D1-D3. injection of osteogenic induction medium (OIM); B1-B14, E1-E3. injection of osteogenic induction medium (OIM) and ADSCs; C1-C14, F1-F3. injection of osteogenic induction medium (OIM), ADSCs and NPY. G. ADSCs-GFP, GFP: green fluorescent protein. H. ADSCs-GFP was localized using laser-scanning confocal microscopy after injection at fracture site four times a week for 7 days. Fig. 4 Imaging and histological evaluation of the therapeutic effects on 21 and 42 days after treatment. A-C. X-ray and micro-computed tomography (micro-CT); D-F. fast green/saf-O staining for histology 42 days after injection. A1-A14,D1-D3. injection of osteogenic induction medium (OIM); B1-B14, E1-E3. injection of osteogenic induction medium (OIM) and ADSCs; C1-C14, F1-F3. injection of osteogenic induction medium (OIM), ADSCs and NPY. G. ADSCs-GFP, GFP: green fluorescent protein. H. ADSCs-GFP was localized using laser-scanning confocal microscopy after injection at fracture site four times a week for 7 days. Bone healing was qualitatively measured by micro-CT expressed as fracture line and callus formation, and assessed by histological examination of decalcified sections. Tibias were longitudinally scanned by micro-CT. At 6 weeks after treatment, there was bigger external bone callus and blurry fracture lines in the NPY-ADSCs group ( Fig. 4 C11-C14 ), while the control group exhibited a smaller callus and still clear fracture lines ( Fig. 4A11-A14 ). The fracture healing in the group in which only ADSCs was injected at the site of tibial fracture was between the vehicle and NPY-ADSCs group ( Fig. 4 B11-B14). Micro-CT quantitative analyses of trabecular BMD and structural parameters of the callus at 6 weeks after treatment also confirmed this result. The trabecular BMD, bone volume per tissue volume (BV/TV), rabecular number (Tb.N), and trabecular thickness (Tb.Th) increased significantly and trabecular separation (TbSp) narrowed in NPY-ADSCs group ( Table 1 ). Table 1 Trabecular bone mineral density and the microscopic structural parameters of the callus of rat tibias at 42 days after treatment in three groups. Table 1 OIM OIM + ADSCs OIM + ADSCs + NPY P value BV/TV (%) 10.1 ± 2.10 25.8 ± 1.05 37.5 ± 1.37 0.001 TbN (mm-1) 1.56 ± 0.10 2.23 ± 0.44 2.57 ± 0.18 0.004 TbTh (mm) 0.069 ± 0.01 0.075 ± 0.03 0.089 ± 0.03 0.010 TbSp (mm) 0.332 ± 0.05 0.224 ± 0.02 0.179 ± 0.02 0.003 TbBMD (mgHA/cm3) 277.5 ± 16.8 348.9 ± 20.0 406.2 ± 35.6 0.001 BV/TV: bone volume per tissue volume; TbN: trabecular number; TbTh: trabecular thickness; TbSp: trabecular separation; TbBMD: trabecular bone mineral density; OIM: osteogenic induction medium; P values are given after multiplicity correction. Trabecular bone mineral density and the microscopic structural parameters of the callus of rat tibias at 42 days after treatment in three groups. BV/TV: bone volume per tissue volume; TbN: trabecular number; TbTh: trabecular thickness; TbSp: trabecular separation; TbBMD: trabecular bone mineral density; OIM: osteogenic induction medium; P values are given after multiplicity correction. Safranin O/fast green staining showed that less new cartilage tissue was found in the vehicle group ( Fig. 4 D) than in the NPY-ADSCs group ( Fig. 4 F) at 6 weeks post-fracture. The amount of new cartilage tissue in the group in which only ADSCs was injected at the site of tibial fracture was between the vehicle and NPY-ADSCs group ( Fig. 4 E). We established the primary culture ADSCs of green fluorescent protein (GFP)-expressing mouse and amplified it in vitro, providing a cytological research platform for the next step. GFP-labeled ADSCs were fusiform and fibroblast-like cells ( Fig. 4 G) and were injected at fracture site four times a week for 7 days. And then we removed the fractured tibia and maked bone tissue section. GFP were localized using laser-scanning confocal microscopy. We showed that green fluorescent ADSCs were observed at the fracture site ( Fig. 4 H). We found that only NPY Y1 receptor was expressed at the ADSCs surface (Agarose gel electrophoresis, Fig. 5 A and Fig. S4 ). Western Blot result confirmed it ( Fig. 5 B and Fig. S5 ). At the existence of NPY, Y1 receptor antagonist (BMS 193885: 10 −9  M) could promote the osteogenetic differentiation of ADSCs, and Y1 receptor agonist (Leu31, Pro34: 2 × 10 −9  M) could partially counteract the promotion effect of osteogenetic differentiation of ADSCs by NPY ( Fig. 5 C–H). At the absence of NPY, Y1 receptor antagonist (BMS 193885: 10 −9  M) could also promote the levels of mineralized nodules formation ( Fig. 5 I), and promote the mRNA level of ALP, OC, and runx2 ( Fig. 5 J). Fig. 5 Effect of neuropeptide Y1 receptor antogonist and angonist on the osteogenetic differentiation. A-B. Only Y1 receptor was expressed at the ADSCs surface (A.agarose electroph oresis; B.Western Blot, full-length gels/blots were presented in Supplementary Fig. S4 and Fig. S5 ); C-H. Effect of Y1 receptor antagonist and angonist on the osteogenetic differentiation of ADSCs (C-H. in the presence of NPY. C-E. ALP staining at day 14; F-H. alizarin red S at day 21). I-J. The effect of Y1 receptor antagonist on the osteogenic related mRNA (ALP, OC, runx2) in the absence of NPY. Fig. 5 Effect of neuropeptide Y1 receptor antogonist and angonist on the osteogenetic differentiation. A-B. Only Y1 receptor was expressed at the ADSCs surface (A.agarose electroph oresis; B.Western Blot, full-length gels/blots were presented in Supplementary Fig. S4 and Fig. S5 ); C-H. Effect of Y1 receptor antagonist and angonist on the osteogenetic differentiation of ADSCs (C-H. in the presence of NPY. C-E. ALP staining at day 14; F-H. alizarin red S at day 21). I-J. The effect of Y1 receptor antagonist on the osteogenic related mRNA (ALP, OC, runx2) in the absence of NPY.

ADSCs of rats were isolated and cultured as previously described [ 11 ]. Briefly, female Sprague Dawley rats' inguinal subcutaneous white adipose tissues were obtained before fracture. The ADSCs were cultured in modified Eagle's media (DMEM, Gibco) containing 10 % fetal bovine serum (FBS), 1 % penicillin and streptomycin (Gibco) at 37 °C in 5 % CO2. The following experiments were conducted using the third passage of cells grown to 80 %–90 % confluency using 0.25 % trypsin-EDTA (Gibco). Experiments were repeated at least 3 times. The morphology of ADSCs was observed via microscopy (Leica, DMI 4000, Germany) and the immunophenotypes of ADSCs (CD44/CD45-) were characterized by LSR II flow cytometry (BD Bioscience). The data were analyzed by Flowjo X 10.0 (Tree Star Inc.). All experiments were conducted in triplicate. ADSCs’ osteogenic differentiation were measured via Alkaline Phosphatase (ALP) staining and Alizarin Red staining (Solarbio, China) [ 12 ]. For identification of ADSCs differentiation, cells were induced for 14 days and 21 days with different treatment. Then cells were fixed in 0.4 % formaldehyde (Beyotime Institute of Biotechnology, China) for 5 min, washed with PBS and deionized water, and stained with 2 % Alizarin Red solution for 10 min, then rinsed with distilled water. ALP activity was evaluated using an ALP staining kit. The osteogenic differentiation and mineralization level of ADSCs was evaluated according to the intensity of ALP and Alizarin Red staining. To determine the effects of NPY (10 −4  M, 10 −6  M, 10 −8  M, 10 −10  M and 10 −12  M) on osteogenic differentiation, ADSCs were cultured in osteogenic media (growth culture media supplemented with 20 mM β-glycerophosphate, Sigma, 50 mM L-ascorbic acid-2-phosphate, Sigma, and 1 nM dexamethasone, Sigma) for 14–21 days. The media were refreshed three times a week during osteogenic differentiation. Y1 receptor antagonist (BMS 193885, R&D, 1 × 10 −9  M), Y1 receptor agonist (Leu31, Pro34, R&D, 2 × 10 −9  M). Modified ferro O- solid green cartilage staining was operated using the Staining kit (Solarbio, China). Fifty-seven female Sprague Dawley rats, aged 6 months with the body weight of 270 ± 30 g, were included in this study. Fifteen rats were used to study the effect and mechanism of NPY on the osteogenic differentiation of rats' ADSCs in vitro. Forty-two rats were used for operation. Four green fluorescent proteins (GFP) mouses (Jackson lab, stock number 006567) aged 2 months, two female and two male, were included in this study. Every four animals were kept in one cage with climate-controlled conditions (25 °C, 55 % humidity, 12 h of light alternating with 12 h of darkness). Free access to tap water and standard laboratory diet were permitted [ 13 ]. All the animal experiments were conducted in accordance with international standards on animal welfare as well as being compliant with the Animal Research Committee of the university. For animal welfare, animals were monitored on a daily basis and examined for following indications: body temperature, body weight changes, clinical signs, water and food consumption, and behavior. After bilateral ovariectomy (n = 39) or sham operation (n = 3) according to a previous report [ 14 ], 3 months were allowed to pass before fracture surgery for the establishment of standard osteoporotic animal models [ 15 ]. Then three randomly selected ovariectomized (OVX) rats and the three sham-operated ones were euthanized for bone mineral density (BMD) and body weight, and the uterus was harvested for comparison of size and uterine weight to confirm the establishment of osteoporotic animal model. Next, the remaining 36 OVX rats were then used for the tibial fracture model. Briefly, rats were generally anesthetized before fracture. To obtain a reproducible fracture in the rats' tibia, we used our own design of fracture modeling support, which had been granted utility model patent. A 1-mm-diameter Kirschner's wire was inserted into the tibia through the patellar tendon, then the bottom third of tibial fracture was conducted using the fracture modeling support. For the first 3 days post-operatively, pain levels of the rats were controlled by injecting buprenorphine every day. All efforts were made to help rats suffer the least and all surgical procedures above-mentioned were performed in a sterile environment. All rats received a postoperative X-ray on the following day to confirm the success of the transverse tibial fractures. Three rats were excluded due to comminuted fracture patterns, leaving 33 rats in the modeling group. After fracture operation, all animals were randomly divided into four groups, and the pharmaceutical interventions commenced from the second postoperative day through the following injections at fracture sites four times a week: vehicle (osteogenic differentiation fluid) (group ODF-vehicle, n = 10); ADSCs plus osteogenic differentiation fluid (ADSCs derived from the osteoporotic fracture rats, 2 × 10 10  cells/ml, group ADSCs, n = 10); NPY (10 −8  M) plus ADSCs (derived from the osteoporotic fracture rats, 2 × 10 10  cells/ml) and osteogenic differentiation fluid (group. NPY and ADSCs, n = 10); ADSCs derived from GFP mouse plus osteogenic differentiation fluid (group GFP, n = 3). All rats received a volume of 20 μl/side at an infusion rate of 5 μl/min. During treatment, the body weight of the animals was measured weekly. All experimental rats survived without any complications, including infections. Five rats from the three groups (group ODF-vehicle, group ADSCs, group NPY and ADSCs) were randomly sacrificed at three weeks and six weeks after the surgery. Tibias were removed and fixed in 4 % paraformaldehyde for 24 h. Fracture callus was evaluated using a micro-CT system (Quantum GX, PerkinElmer, America) at a resolution of 72 μm with 90 kV and 80 μA. We constructed 3-dimensional structures. After micro-CT scanning, three rats’ tibias from the three groups (group ODF-vehicle, group ADSCs, group NPY and ADSCs) were randomly decalcified with 10 % EDTA solution for 3 weeks and were subsequently embedded in paraffin. Longitudinal, sagittal thin (7 μm) sections were sliced by a Leica RM2235 microtome (Leica Biosystems, Nussloch, Eisfeld, Germany). Sections were then mounted on glass slides, and stained with Safarin-O/Fast green method to allow identification of bone and cartilage. ADSCs, derived from GFP mouse plus osteogenic differentiation fluid (group GFP), were injected at rats' fracture sites four times a week for one week. The three rats’ tibias were then sectioned longitudinally through the fracture sites using a hard tissue saw microtome (Buehler 699-ISF-6531, China) to produce specimens that were ∼100–120 mm thick. Sections were mounted on glass slides, and observed using the laser scanning confocal microscope (LSCM, German Zeiss lsm 780) to allow identification if the ADSCs migrated to fracture were involved in fracture healing. The cultured cells were lysed with RIPA lysis and extraction buffer (KeyGEN Biotechnology, China) to acquire bone-related proteins such as alkaline phosphatase (ALP), runt-related transcription factor 2 (runx2), osterix, bone morphogenetic protein 2 (BMP2) and osteocalcin (OC). The concentration of these proteins was determined via the bicinchoninic acid (BCA) protein quantification kit (Beyotime, China). And Western blot was conducted as previously described [ 16 ]. The β-catenin was used as an internal control. The primary antibodies used in the experiments were anti-ALP (1:1000; Abcam, USA), anti-runx2 (1:1000; Abcam, USA), anti-BMP2 (1:1000; Abcam, USA), anti-OC (1:1000; Abcam, USA) and anti-β-catenin (1:1000; Abcam, USA). Total RNA was isolated from cells induced for 14 and 21 days using Trizol reagent (Thermo Fisher Scientific, USA). A Reverse Transcription System (TaKaRa, Japan) was used to reverse-transcribe messenger RNA to complementary DNA (cDNA) and qRT-PCR was performed with SYBR Green PCR master mix (TaKaRa, Japan) on an ABI 7900 fast real-time PCR system (Applied Biosystems, Carlsbad, USA). β-catenin was used as an internal control to normalize the expression levels of mRNA. The relative expression levels were then calculated using the 2−ΔΔCT method. The primers for β-catenin, runx2, osterix and OC were purchased from Sangon (Shanghai, China). All studies were randomized to reduce the influence of confounding factors and the randomization sequence was generated by a random number table. All independent experiments were performed in triplicate and all quantitative data were expressed as mean ± SD. Statistical analysis was performed via GraphPad software 7.0 and SPSS 20.0. Two-tailed Student's t-test was used for comparisons between two groups and One-Way Analysis of Variance (ANOVA) with Bonferroni post hoc test were used to calculate the P -values for multiple comparisons. A value of P  < 0.05 was considered as statistically significant and represented as the symbols “∗“. P  < 0.01 was represented as the symbols “∗∗“. P  < 0.001 was represented as the symbols “∗∗∗“.

The findings of this study combined with those in previous reports suggest that NPY may play a role in the regulation of skeletal homeostasis, probably through Y1R. This knowledge provides a platform for the development of novel therapies for bone regeneration with endogenous ADSCs.

Bone grafting is widely used in hospitals to repair damaged, aging, or diseased bone tissue. Grafting of bone facilitates bone healing by stimulating osteogenesis (i.e., bone generation) at the site of injury, but this can only be achieved when augmentation includes cells capable of forming bone [ 17 ]. This procedure is the safest and most effective since it uses the patient's own cells (to facilitate osteogenesis) and proteins (to facilitate osteoinduction), as well as providing a framework for the formation of new bone (osteoconduction) [ 17 , 18 ]. However, bone autograft is limited in quantity (about 20 cm 3 ) and its harvesting (e.g., from the iliac crest) requires an additional surgery, which is often painful and causes complications [ 17 , 18 ]. The next solution is allograft bone directly obtained from tissue banks (fresh-frozen) or prepared to be conserved (dried or lyophilized). This solution does not contain viable cells and some matrix proteins are disrupted by virus-inactivation treatments and the freezing procedure; thus, it only ensures osteogenesis induction properties. Moreover, allograft bone may transfer disease or lead to immunological rejections [ 17 , 19 ]. In addition to bone grafts, substitutes and their reinforcer with growth factors and synthesis strategies, cell therapies have been gradually proposed to evolved into new osteoinductive and osteogenic medium that can be used safely and effectively competing with currently available criteria. MSC-based cell therapy is an interesting alternative. MSCs are non-haematopoietic stromal stem cells that can be derived from a variety of tissues, including periosteum, fat, bone marrow, muscle, umbilical cord blood, peripheral circulation, skin and dental tissues. In addition to self-replication, they are able to differentiate into and contribute to mesenchymal tissues, including bone, ligaments, cartilage, muscles, tendons, and fat tissue. Bone marrow MSCs are currently the most appropriate cells for inducing bone repair, because they have strong osteogenic potential and can be obtained by culturing iliac crest aspirates. However, the number of bone marrow MSC is limited and the collection requires additional surgical intervention and often results in pain and complications [ 17 ]. Patients with fracture healing received bone marrow MSC cells at an average concentration of 1500 per mL, with 54, 000 in a total volume of 20 mL [ 20 ]. Adipose derived mesenchymal stem cells (ADSCs) have osteogenic potential and can be derived from a wide range of sources to meet the sufficient concentration of mesenchymal stem cells required for osteoporotic fracture healing. ADSCs could be obtained from autologous subcutaneous adipose tissue. In the course of MSC-based cell therapy, how to enhance osteogenic differentiation of ADSCs is the most important problem to be solved. Bone remodeling is mainly regulated by endocrine and paracrine, and bone homeostasis is also controlled by central and peripheral nerves, the most important of which is the NPY system. NPY is known to be expressed in bone and adipose tissue and acts as a neurotransmitter to regulate bone metabolism [ 4 , [21] , [22] , [23] , [24] , [25] ]. Our study shows that NPY promotes osteogenic differentiation of ADSCs in rats. This was based on the observation that NPY treatment led to upregulation of osteogenic genes and typical morphological changes after 14 and 21 days of osteogenic differentiation. The effect of NPY on osteogenic differentiation of BMSCs has been supported by previous studies. Liu et al. found that NPY had a concentration-dependent positive effect on osteogenic differentiation of BMSCs, while Ma et al. found that NPY and BMP2 increased osteoblast activity by increasing inter-cellular communication in the gap junction [ 26 , 27 ]. In our study, ADSCs were obtained from rats’ autologous adipose tissue. We added the NPY into the osteoblastic differentiation medium and injected the infusions at fracture sites four times a week. Our results showed that autologous ADSCs with exogenous NPY transplantation could stimulate osteoporotic fracture healing in vivo. We further used GFP-tagged murine ADSCs transplantation and found the participation of exogenous ADSCs during fracture healing. Previous studies have shown that serum NPY in SD rats increased significantly after fracture healing and reached a peak at the 8th week of fracture healing [ 9 ]. NPY inhibitors can inhibit fracture healing [ 9 ]. The possible function of the peripheral NPY system in local bone remodeling was also highlighted in another study, which showed regeneration of NPY fibers in callus and braided bone on day 7 of tibial fracture in SD rats [ 28 ]. Our findings add to a growing body of evidence that exogenous NPY combined with autologous ADSCs could promotes osteoporotic fracture healing. The study holds potential significance for understanding and developing fracture treatment methods for osteoporosis patients, particularly postmenopausal women. NPY acts through several different receptor subtypes (Y1, Y2, Y4, Y5, and Y6) [ 29 ]. NPY receptor subtypes are limited to tissue-specific expression and explain the different roles of NPY in different tissues [ 29 ]. Our study showed that only Y1R was expressed on the surface of ADSCs. Furthermore, these cells exhibit a reduction in calcium deposition in extracellular matrix mediated via Y1R agonist in the presence or absence of NPY. In contrasts, it was found that mineralized nodules were more obvious after the addition of Y1R antagonist. Another previous study showed that NPY was a negative regulator of Y1 receptor expression, and exogenous NPY down-regulates Y1R expression on the surface of MC3T3 cells [ 30 ]. Functional analysis also revealed the osteogenic potential of NPY, and osteoblast phenotypic markers were significantly enhanced in osteoblasts stimulated by NPY, which may be related to the down-regulation of Y1 receptor [ 30 ]. NPY receptor function is regulated by multiple mechanisms, including receptor internalization/desensitization and recycling/re-sensitization [ 31 ]. The C-terminal of the Y1 receptor plays a role in desensitization and internalization [ 32 ]. Previous studies had pointed out that Y1 receptor was easily desensitized after prolonged exposure to agonists, and the response was rapidly weakened [ [32] , [33] , [34] ]. Studies using radio-labeled agonists [ 35 ] or GFP-labeled Y1 receptors [ 36 ] had revealed rapid cycling of the receptors [ 32 , 36 , 37 ]. In contrast, the Y2 receptor also binds NPY and PYY with high affinity, with less desensitization and endocytosis [ 36 , 37 ].

Our study has some limitations. First, we recognized the limitations of simulating human clinical disease using animal models and our current model also has limitations and requires validation in a large animal model. Second, we only studied the pro-osteogenic effects of NPY on ADSCs, and the effects of NPY on bone-resorbing osteoclasts and chondrogenic differentiation of ADSCs should be explored in the future. Third, our findings provide some tentative initial evidence that exogenous NPY promotes osteogenic differentiation via down-regulating Y1R expression on the surface of ADSCs. We acknowledge that future work about additional in-depth mechanism study is required, particularly on the unique signaling pathway and molecular mechanism of NPY.

In recent years, the view of bone remodeling as primarily controlled by endocrine, paracrine and mechanical processes has expanded to incorporate the importance of central and peripheral neuronal signals [ 1 ]. Neuropeptide Y (NPY) is an important signaling pathway that is widely expressed in the central nervous system (CNS) and peripheral nervous system (PNS) [ 1 ]. NPY regulates appetite and energy balance in the central nervous system [ 2 ] and plays a protective role against chronic bone loss-induced stress [ 3 ]. It has been reported that NPY is expressed in several cell types, including osteoblasts and adipocytes [ 4 ], as well as being important in regulating bone mass throughout the body [ 1 , 5 ]. As compared to other sources, adipose-derived stem cells (ADSCs) are one of the most promising cells for regenerative medicine, due to they possess a higher differentiation potential and are easily propagated [ 6 ]. As mesenchymal stem cells (MSCs), ADSCs are capable of maintaining their undifferentiated state, controlled by the stemness related genes, as well as differentiating [ 6 ]. ADSCs can differentiate into osteogenic cells given the appropriate conditions [ 7 ]. This process is regulated by numerous osteogenesis factors including runt-related transcription factor 2 (runx2), osterix, bone morphogenetic protein (BMP), osteocalcin (OC), and alkaline phosphatase (ALP) [ 7 ]. As the master transcriptional factor of osteogenesis, runx2 plays an essential role in osteogenic differentiation and normal skeletal development [ 7 ]. Patients with osteoporosis are more likely to suffer from fractures, which can cause significant morbidity and mortality. Moreover, osteoporotic fractures are difficult to treat because of their decreased bone strength and suboptimal healing capacity, leading to both fixation failures and non-unions [ 8 ]. By combining synthetic bone graft substitutes with bioactive molecules, such as growth factors, peptides, and small molecules, bone precursor cells can be delivered more efficiently, resulting in better bone formation and metabolism [ 8 ]. It has been demonstrated in murine studies that both NPY ligands and receptors are active regulators of bone mass and osteoblast activity as well as integrating with mechanical signals during local loading [ 1 ]. As fracture healing progressed, serum levels of NPY increased significantly, reaching a peak in the 8th week [ 9 ]. In our previous study, we found that NPY could regulate the differentiation of primary cultured human ADSCs [ 10 ]. In the present study, we hypothesize that NPY combined with ADSCs may facilitate clinical translation and provide novel insights into bone transplantation in the future.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Hong Liu, Houde Zhou reports a relationship with the 10.13039/501100004735 Natural Science Foundation of Hunan Province or China that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The data underlying this article are available in the article and in its online supplementary material.