Icariin accelerates cartilage defect repair by promoting chondrogenic differentiation of BMSCs under conditions of oxygen‐glucose deprivation

Abstract This study explored the role played by combined ICA and bone mesenchymal stem cells (BMSCs) in repairing rabbit knee cartilage defects. Firstly, rabbit BMSCs were isolated and used to construct an in vitro cellular model of oxygen‐glucose deprivation/reoxygenation (OGD/R). Subsequently, ICA processing, Alcian blue staining, immunofluorescence and Western blot studies were performed to evaluate the ability of BMSCs to display signs of chondrogenic differentiation. Furthermore, a rabbit knee cartilage injury model was established in vivo. International Cartilage Repair Society (ICRS) macroscopic evaluations, H&E, Alcian blue and EdU staining, as well as immunohistochemistry, were analysed cartilage repair and pathological condition of the knee cartilage tissue. Our in vitro results showed that ICA promoted the chondrogenic differentiation of BMSCs, as well as aggrecan (AGR), bone morphogenetic protein 2 (BMP2) and COL2A1 protein expression in BMSCs. In vivo experiments showed that rabbits in the BMSCs or ICA treatment group had higher ICRS scores and displayed a better restoration of cartilage‐like tissue and chondrocyte expression on the surface of their cartilage defects. In conclusion, ICA or BMSCs alone could repair rabbit knee cartilage damage, and combined treatment with ICA and BMSCs showed a better ability to repair rabbit knee cartilage damage.

tissue repair. 8 Research has shown that mesenchymal stem cells extracted and expanded from bone marrow can promote the regeneration of bone and cartilage, 9,10 and immune regulation, 11 which prevents further cartilage damage. Bone marrow mesenchymal stem cells (BMSCs) have a paracrine function that regulates surrounding cells and are beneficial to the synthesis of cartilage matrix. 12 A recent study showed that BMSC-assisted injectable Collagen I hydrogel can regenerate a cartilage defect and remould cartilage homeostasis. 13 However, when BMSCs are transplanted into a region of ischaemic and hypoxia tissue, the survival rate of the BMSCs is reduced, and their repair ability is also impaired. 14 Therefore, enhancing the tolerance of BMSCs to ischaemia and hypoxia, as well as improving graft survival are essential for achieving cartilage repair.
Icariin (ICA) is a flavonoid glycoside compound and effective pharmacological ingredient found in the traditional Chinese medicine Epimedium. 15 ICA has anti-osteoporosis, anti-inflammation, antitumour and anti-oxidative stress effects and also helps to protect cardiovascular function. 16 ICA has been widely used for treating osteoporosis 17 and also in experimental research on osteoblasts 18 and osteoclasts. 19 In addition, ICA can suppress the lipopolysaccharideinduced inflammation of chondrocytes. 20 All these studies suggest the role of ICA on cartilage tissue engineering. The introduction in the previous paragraph has demonstrated the promoting effects of BMSCs on the regeneration of bone and cartilage. However, the therapeutical effects of BMSCs are discounted due to restricted survival time of BMSCs. Interestingly, previous study has shown that ICA can induce the synthesis of alkaline phosphatase and formation of calcified nodules to promote the differentiation of rat BMSCs into osteoblasts. 18 Moreover, ICA has been reported to exert antiapoptosis effect on various cells. [21][22][23] Recent study has also proved that ICA has the potential to induce BMSC proliferation and differentiation into chondrocytes and protect rabbit BMSCs against oxygen-glucose deprivation (OGD)-induced apoptosis. 24

| Cell isolation and culture
New Zealand rabbits (1-month old) were purchased from the animal holding unit of the Fourth Military Medical University. BMSCs were isolated from rabbit bone marrow as previously described. 25,26 In brief, samples of bone marrow were cultured in DMEM (Gibco, Waltham, MA, USA) containing 100 U/ml penicillin/streptomycin, 272 μg/ml L-glutamine and 10% foetal bovine serum (FBS, Gibco).
The medium was changed every three days.

| Stem cell characterization assay
BMSCs isolated from bone marrow were cultured to passage 3 and subsequently cultured for osteogenic, chondrogenic and adipogenic differentiation to explore the differentiation characteristics of stem cells. 24,27 For analysis of osteogenic differentiation, BMSCs were induced and then cultured in osteogenic induction medium at a density of 3 × 10 3 cells/mL for 14 days; after which, they were fixed

| Cellular oxygen and glucose deprivation treatment
BMSCs were cultured in glucose-and serum-free DMEM within an oxygen-free incubator at 37°C for 4 h and then reoxygenated in DMEM medium for use in further experiments. To evaluate the effect of ICA on oxygen-glucose deprivation/reoxygenation (OGD/ R)-induced BMSCs, 0.1, 1 or 10 µM ICA (PureOne Biotechnology, Shanghai, China) was added to the medium at 24 h before OGD/R conditions were applied. The cells were divided into five groups that included a Control, OGD/R, OGD/R + 0.1 µM ICA, OGD/R + 1 µM ICA and OGD/R + 10 µM ICA group. Simultaneously, the cells were also cultured in chondrogenic induction medium for subsequent analysis of their chondrogenic ability by Alcian blue staining.

| Immunofluorescence
Immunostaining was performed to analyse the expression of bone morphogenetic protein 2 (BMP2), aggrecan (AGR) and collagen type II alpha 1 (COL2A1) in BMSCs after 3 days, 7 days and 14 days of incubation in chondrogenic differentiation medium. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.1% Triton X-100 and then blocked with 1% bovine serum albumin in PBS. Next, the cells were incubated with rabbit polyclonal anti-BMP2, AGR and COL2A1 antibodies (Abcam, Cambridge, MA, USA) overnight at 4°C and subsequently incubated with a CM-Dil labelled goat anti-rabbit secondary antibody. In addition, the actin cytoskeleton was stained for 45 min with phalloidin-Atto488 (Sigma-Aldrich), and BMP2 and AGR markers were also stained in the cells. After washing with PBS, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), and staining results were observed under a confocal microscope (Nikon EclipseTS100, Tokyo, Japan).

| DMMB assay of GAG
BMSCs were cultured in glucose-and serum-free DMEM at 37°C for 24 h. After incubating for 24 h, the conditioned medium was collected for GAG synthesis analysis. Conditioned medium was collected from each experimental group, and the presence of GAG released from primary chondrocytes was quantified using dye DMMB.
Culture medium was pretreated with 0.5 units/ml of hyaluronidase at 37 °C for 3 h in order to get rid of exogenous HA in order to remove interference. Digests were mixed with DMMB in 96-well plates and read at 520 nm with Spectra Max 384 Microplate Reader.

| Western blotting
The levels BMP2, AGR and COL2A1 (1:1000, Abcam, Cambridge, USA) protein expression in BMSCs after 3 days, 7 days and 14 days of incubation in chondrogenic differentiation medium were further analysed by Western blotting. BMSCs were lysed in RIPA cell lysis buffer containing a protease inhibitor, and the amount of total protein in each sample was detected using a BCA protein assay kit (Solarbio, Beijing, China). Next, a 50 µg aliquot of total protein from each sample was separated by 12% SDS-PAGE, and the protein bands were transferred onto polyvinyliene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skimmed milk for 2 h at room temperature and then incubated overnight with primary antibodies 4°C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h. Next, the membranes were washed 3 times with TBST and visualized with an enhanced chemiluminescence system (Merck Millipore, Darmstadt, Germany). The signal intensity in the films was analysed with an Alphalmager HP system (Cell Biosciences, Inc., Santa Clara, CA, USA).

| Construction of the articular cartilage injury model
A total of 84 six-month-old New Zealand white rabbits (2.5 kg each) were used in this study. The growth environment temperature of all animals is maintained at 22°C, and the relative humidity is 55%. The sterilized special feed was provided, and the food and water resources were freely obtained. The rabbits were anaesthetized by intravenous injection of 3% pentobarbital (40 mg/kg) supplemented with a subcutaneous injection. Next, each anaesthetized rabbit was fixed on an operating table in the supine position, the hair around the right knee joint was shaved with a razor, and the right knee joint was wiped with iodophor. The right knee joint was covered with a hole towel to expose the knee surgery area. The knee joint was bent along the inner side of the patellar ligament, and a 3.0-4.0 cm incision was made on the inner side of the knee joint patellar ligament. Next, the patella was pushed to the outer side to expose the femoral trochlear. A hand drill was used to create a cartilage defect on the trochlear surface of the femur. The trochlear surface of the femur was positioned with a 3 mm drill bit, and a hole with a diameter of 3 mm and a depth of 4 mm was drilled.
PBS was used to wash away tissue debris and blood clots. Finally, the patella was reset, and the wound was sutured and disinfected with iodophor. Penicillin (800,000 U) was continuously injected into the breech muscle each day for 3 consecutive days after the operation.
This protocol for this study was approved by the Institute Animal Care and Use Committee of Guangzhou Hospital of Integrated Traditional and Western Medicine (Guangzhou, Guangdong, China). This study followed the ARRIVE guidelines.

| ICA and BMSC processing
The above animal models were randomly divided into 4 groups with 7 rats in each group. These models included an Operation (Operation), Operation + BMSCs (BMSCs), Operation + ICA (ICA) and Operation + ICA + BMSCs (ICA + BMSCs) group. The specific treatment process was as follows. 8 × 10 5 U of penicillin was injected intramuscularly into the breech for 3 days after modelling. In the Operation group, the joint cavity was injected with an equal volume of saline (1 ml). In the BMSCs group, the joint cavity of the rabbit was injected with 1 ml of rabbit BMSCs (1 × 10 7 cells) in the first two treatments. In the third treatment, the joint cavity was injected with 1 mL of rabbit BMSCs (1 × 10 7 cells) that had been incubated with 10 μM EdU for 48 h in advance. In the ICA group, 1 ml of 10 μM ICA was injected into the ear vein for a total of three administrations. In the ICA + BMSCs group, the joint cavity of the rabbit was injected with 1 ml of rabbit BMSCs (1 × 10 7 cells) in the first two treatments, and in the third treatment, the joint cavity was injected with 1 ml of rabbit BMSCs (1 × 10 7 cells) that had been incubated for 72 h with 10 μM ICA in advance and also incubated with 10 μM EdU for 48 h.
Then, after the last treatment, rabbits were observed for 4 weeks, 8 weeks and 12 weeks respectively (Table 1).

| ICRS
At 4, 8 and 12 weeks, respectively, 5 rabbits in each group were sacrificed by anaesthesia. The whole knees of the rabbits were dissected, and the distal femur was removed. Cartilage damage was assessed based on the International Cartilage Repair Society (ICRS) gross morphology assessment scale for cartilage repair as shown in Table 2. And three scientists performed the scoring, and it was treatment blinded.

| Pathological analysis
The lower ends of the femurs were fixed with paraformaldehyde for 1 day and then decalcified in 10% EDTA (pH 7.3) for 21 days. were also observed under an optical microscope.

| Immunohistochemistry
After tissue decalcification and paraffin embedding, immunohistochemistry was performed. 29 The sections were incubated with rabbit anti-COL2A1 antibody at 4°C overnight and subsequently incubated with the secondary goat anti-rabbit antibody labelled with horseradish peroxidase for 30 min at 37°C. The colour reaction was developed with 3, 3′-diaminodenzidine, and the tissue sections were counter-stained with haematoxylin. The prepared sections were observed under an optical microscope.

| Cartilage repair ability analysis
In order to further analyse the repair of cartilage in the cartilage damage area, EdU staining was performed to analyse cell prolif-

| Statistical analysis
All experiments were repeated three times, and the data were analysed using GraphPad Prism 8.0.2 software. Differences between groups were analysed by one-way or two-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test for multiple comparisons. A p-value < .05 was considered to be statistically significant.

Times 1 w 3 d (once a day) 1 w (once a week) 1 w (once a week) 1 w (once a week) Group
Operation (21) Adaptive feeding Modelling + Penicillin

| ICA induced glycosaminoglycan expression in BMSCs
Based on characteristics of the isolated BMSCs, we selected the third-  Figure 1 (Figure 1C).

| ICA induced the chondrogenic differentiation of BMSCs
Based on the above results, we further analysed the ability of ICA to induce cartilage formation by BMSCs. We detected the contents of AGR and COL2A1 in chondrocyte extracellular matrix, 30 as well as the levels of BMP2, which is one of the most effective factors for inducing the chondrogenic differentiation of mesenchymal stem cells. 31 Immunofluorescence results showed that the levels of BMP2, AGR, COL2A1 and actin cytoskeleton protein expression and cell proliferation in the OGD/R group were all lower than those in the Control group, indicating that the proliferative and differentiation abilities of BMSCs in the OGD/R group had been reduced (Figures 2 and 3A). This could be explained by the fact that hypoxia, sugar-free and serum-free treatment had reduced the proliferation and chondrogenic differen-  shown in Figure 2 (Figures 2 and 3A). In addition, the Western blot results showed that the levels of BMP2, AGR and COL2A1 protein expression in the OGD/R group were significantly reduced when compared with expression in the Control group at 3, 7 and 14 days

| ICA and BMSCs combined to repair knee cartilage damage in vivo
Next, ICRS criteria were used to evaluate the repair level of damaged cartilage tissue at 4, 8 and 12 weeks after BMSC and ICA treatment respectively. The mean ICRS score at 4 weeks in the Operation group was 1.4 ± 0.55, which was lower than the mean ICRS score at 4 weeks in both the ICA group (3.2 ± 0.84) and BMSCs group (3.0 ± 0.71), indicating that the cartilage in the rabbit knee joint was severely abnormal, and had not obviously improved after BMSC or ICA treatment ( Figure 3D).
However, the mean ICRS score in the BMSC + ICA group at 4 weeks was 5.8 ± 1.3, which was significantly higher than the mean ICRS score in both the BMSCs group (p < .001) and ICA group (p < .001), suggesting that the extremely damaged cartilage was partially repaired after BMSC plus ICA treatment at 4 weeks ( Figure 3D). The mean ICRS scores at 8 weeks in the Operation, BMSCs, ICA and BMSCs + ICA groups were 3.0

| ICA and BMSCs combined to affect the pathological changes and proliferation of damaged cartilage tissue
We also observed the pathological features of damaged cartilage  protective role in preventing cartilage destruction, but also promotes chondrogenic differentiation of BMSCs, which further enhance the recovery of cartilage damage. However, the potential mechanism for that recovery from cartilage damage remains to be explored.

| CON CLUS ION
In conclusion, treatment with ICA or BMSCs alone was shown to repair rabbit knee cartilage damage. Moreover, treatment with a combination of ICA and BMSCs demonstrated an even better ability to repair rabbit knee cartilage defects, which provides a certain basis for the treatment of osteoarthritis.

CO N S E NT FO R PU B LI C ATI O N
Consent for publication was obtained from all authors.

CO N FLI C T S O F I NTE R E S T
All authors declare having no conflicts of interest related to this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.