Effects of icariin on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells

doi:10.21203/rs.3.rs-67547/v1

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Effects of icariin on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells

https://doi.org/10.21203/rs.3.rs-67547/v1

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published 02 Dec, 2020

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Background

Tissue engineering technology has been applied extensively for clinical research and human amnion mesenchymal stem cells (hAMSCs) could cause mesenchymal stem cells to differentiate into the bone tissue. However, it is necessary to develop and identify the safer appropriate amount of osteogenic inducer. The objective of this study is to investigate the effect of icariin (ICA) on the proliferation and osteogenic differentiation of hAMSCs.

Methods

The morphology and phenotype of hAMSCs were discovered by flow cytometry and immunocytochemical staining. The osteogenic differentiation of hAMSCs under the influence of different concentrations of ICA were assessed by Alkaline phosphatase (ALP) activity substrate assay and Alizarin red Staining.

Results

MTT assay revealed that the hAMSCs pretreated with ICA exhibited increased proliferation when compared with the control group, and the most optimum concentration of ICA was 1 × 10^− 6 mol/L. The combined analysis of ALP activity and ARS staining showed that ICA could significantly promote the osteogenic differentiation of hAMSCs, and the effect was most significant when the concentration of ICA was 1 × 10^− 6 mol/L.

Conclusion

All above results implied that ICA could significantly increase proliferation and enhanced the osteogenic differentiation of hAMSCs, especially when the concentration of ICA was 1 × 10^− 6 mol/L.

Surgery

Orthopedics

Human amniotic mesenchymal stem cell

Icariin

Proliferation

Osteogenic differentiation

Bone defects/losses is a serious problem in orthopedics due to low healing rate using traditional treatment, and increases patients' pain and heavily families burdening [1, 2]. Over the last two decades, tissue engineering technology has been applied extensively for clinical research. For example, stem cell-based tissue regeneration showed certain curative effects as a novel curative therapy for bone diseases. However, methods for collection, isolation, and maintenance of stem cells in vitro is still a challenge of stem cell-based clinical healing for bone disorder, which is also disadvantageous to reduce operation time [3]. Moreover, it is still worry whether stem cell would differentiate into bone-forming osteoblasts. Although the osteogenic inducers such as bone morphogenetic proteins (BMP) and vascular endothelial growth factor (VEGF) are important factors for healing of bone defects/losses, there are still some disadvantages for low differentiation efficiency, complicated formulas and high cost [4]. Therefore, the key point is to identify safe and effective osteogenic inducers to improve stem cell-based therapy for bone regeneration.

Previous findings showed that some drugs include statins [5], isoflavone derivatives [6, 7] and TAK-778 [8] could regulate the balance in bone formation between osteoblastic bone formation and osteoclastic bone resorption [9]. Recently, many traditional Chinese medicines were used to fractured bone healing, and some natural small molecule compounds isolated from them were confirmed as effective osteogenic inducers. Icarrin (ICA), as the main active compound of Epimedium pubescens, was reportedly used to cure bone diseases in ancient China [10]. And it could promote agent in cartilage tissue engineering [11] and repair of articular cartilage defects in rabbit knees [12]. It has been found the beneficial effects of ICA is to exert inducing function on the osteogenic differentiation [13] and stimulates the bone marrow-derived mesenchymal stem cells (BMSCs) proliferation in stem cell therapy [14]. Therefore, ICA is expected to be a safe and effective osteoinductive active factor for clinical bone regeneration.

Human amniotic mesenchymal stem cells (hAMSCs), derived from ditched amniotic membrane (AM) [15, 16], are a center of attention in mesenchymal stem cells (MSCs) for bone stem cell-based tissue engineering and regenerative medicine (TE&RM) use owing to their superior properties [17]. HAMSCs has unique advantages for bone regeneration in a noninvasive way, immunomodulatory properties with characteristics of possess weak anti-inflammatory, high differentiation and without ethical controversy [18]. In addition, previous studies found that hAMSCs have been successfully differentiated into osteoblasts using the classic tri-component osteogenic medium and promote bone regeneration in vivo and in vitro [19]. In this study, we aim at make clear effects of icariin on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells.

Cell Isolation, cultivation and identification of hAMSCs

The placental amniotic tissue was collected from normal pregnant women undergoing full-term cesarean section after gaining the informed consent of the pregnant women or her relatives. After removing residual blood under sterile conditions, the amniotic tissue was cut into small pieces of 1–2 cm² and then separated and repeatedly rinsed using phosphate-buffered saline (PBS) containing 100 μg/ml streptomycin and 100 U/ml penicillin (Beyotime, China). Amnion fragments were collected in a 50 mL centrifuge tube and digested for 35 minutes at 37°C in a double volume of digestion solution I (0.05 % trypsin containing 0.02% EDTA-2Na). After centrifugation at 180 rpm for 2 hours at 37°C, the remaining amnion in the centrifuge tube was filtered through a filter screen and digested by a double volume of digestion solution II (0.75 mg/mL collagenase II containing 0.075 mg/mL DNase I) to acquire hAMSCs. The sample was shaken at a speed of 180r/min and digested for approximately 2 hours at 37°C. After filter and centrifuge, the resultant hAMSCs were transferred to low-glucose (LG)-DMEM complete medium, supplemented with 10 percent heat-inactivated fetal bovine serum (FBS) to rest the cell suspension in a humid atmosphere of 5% CO₂ for 5 minutes at 37°C to precipitate hAMSC (passage 0, P0).

The resultant hAMSCs (passage 0, P0) were collected in a 50 mL centrifuge tube and digested for 3 minutes at 37°C in a double volume of digestion solution I (0.05 % trypsin containing 0.02% EDTA-2Na). When the cells reached 80% confluence, they were digested and subcultured into flasks. Cells from passage 3 (P3) were used for further analysis in this study. The cell morphology was constantly observed under a light microscope (Olympus, Tokyo, Japan). In addition, these hAMSCs were analyzed using the immunocytochemical staining method according to the protocols as described in a previous study [20].

For the phenotypic characterization of hAMSCs, P3 hAMSCs were trypsinized and subsequently washed with D-PBS containing 0.1% BSA, adjusted to a density of 1.5×10⁶ cells/mL, and then incubated with and HLA-DR for 25 minutes in the dark. After washing again with D-PBS containing 0.1% BSA, the cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. Finally, the labeled cells were analyzed using the flow cytometry system (FACS Calibur, Becton Dickinson, USA) and CellQuest software (BD, NJ, USA) after fixation with 1% paraformaldehyde.

Cell proliferation assay

This experiment was divided into three groups: blank group (no cells and no ICA), control group (with cells but no ICA) and ICA group (with cells and ICA). The P3 hAMSCs were seeded at a density of 1×10⁴ cells/ml in 96-well plates. After incubation for 24 hours, the medium was changed to ICA-containing media at a concentration of 0 (blank and control), 1×10^-4 (ICA-1), 1×10^-5 (ICA-2), 1×10^-6 (ICA-3), 1×10^-7 (ICA-4) and 1×10^-8 (ICA-5) mol/L accordingly. Cells were incubated at 37°C in a 5% CO₂incubator for 72 hours, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37°C in a 5% CO₂ incubator for 4 hours. Later, the medium was discarded, and 150 μL of dimethylsulfoxide (DMSO) was added to each well. After incubate at 37°C for 15 minutes, the OD value of each well was determined at a wavelength of 490 nm by a microplate reader (MultiskanTM GO, ThermoFisher, Waltham, MA, USA).

Osteogenic and chondrogenic differentiation of hAMSCs in vitro

The P3 hAMSCs were seeded at a density of 1×10⁵ cells/ml in 96-well plates in LG-DMEM culture medium under sterile conditions. After 24 hours of incubation, the medium was replaced with LG-DMEM culture medium with 10% FBS. The experiment was divided into 5 groups according to the different substances added to the medium, namely the classic group (100nmol/L dexamethasone (Sigma, SL, USA), 50 mg/L vitamin C (Solarbio, Beijing, China), and 10mmol/L β-glycerophosphate (Solarbio, Beijing, China)), the blank group (without additives), ICA-1 group (1×10^-4 mol/L ICA), ICA-2 group (1×10^-5 mol/L ICA), ICA-3 group (1×10^-6 mol/L ICA). Cells were incubated at 37°C in a 5% CO₂ incubator for 72 hours, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37°C in a 5% CO₂ incubator for 4 hours. The culture medium was changed every 3 days, and the intervention was continuously induced for 21 days. Meanwhile, during the induction period, the cell morphology of the five periods of 1 d, 3 d, 7 d, 14 d, 21 d were photographed for morphological observation, and samples were preserved. ALP (alkaline phosphatase) was extracted and detected with the ALP assay kit (AnaSpec, San Jose, CA) as directed by the manufacturer. The cells on the 21st day were stained with Alizarin red staining (ARS), and statistical analysis was performed based on the number of stained calcium nodules.

Statistical analysis

Experimental data were expressed as mean±standard deviation (SD) and analyzed using SPSS 19.0 statistical software. One-way analysis of variance was used for comparison between groups, and the rate comparison was performed by χ² test. P < 0.05 was considered as statistically significant.

Morphology and phenotype of hAMSCs

The microscopic examination of P0 hAMSCs cultured in vitro revealed a typical adherent growth state (Fig. 1A). After 48 hours of primary culture, the majority of the adherent cells stretched and became spindle or short-rod shaped. With the increase of passage number, small quantities of epithelial cells and dead cells disappeared rapidly. When the hAMSCs were sub-cultured to the 3nd passage, they displayed a fbroblast-like morphology in radial or whirlpool patterns (Fig. 1A).

Flow cytometry revealed that hAMSCs highly expressed MSC surface markers CD29, CD44, CD73 and CD166 (Fig. 1B). On the other hand, very low expressions of cell surface markers belonging to CD34 and CD45 were showed (Fig. 1B). ICC staining indicated that hAMSCs also highly expressed mesenchymal cell marker vimentin, which proved that hAMSCs were of human origin and a member of the MSC family (Fig. 1C).

Effect of different concentrations of ICA on the proliferation of hAMSCs

The stimulation effect of ICA on the proliferation of hAMSCs during 3 days of culture was evaluated at various concentrations (1 × 10^− 4 mol/L, 1 × 10^− 5 mol/L, 1 × 10^− 6 mol/L, 1 × 10^− 7 mol/L, 1 × 10^− 8 mol/L). OD absorbance values of samples cultured for different times are measured to evaluate the proliferation kinetics of osteoblast-like cells. MTT assay revealed that the hAMSCs pretreated with ICA exhibited increased proliferation when compared with the control group. Meanwhile, in the presence of ICA, the proliferation fold of hAMSCs increases first and then decreases with the increase of ICA concentration. For example, ICA increased the proliferation from 0.524 ± 0.033-fold in the control group to 0.728 ± 0.048- (p < 0.05), 0.719 ± 0.044- (p < 0.05), 0.688 ± 0.057- (p < 0.05) and 0.659 ± 0.041-fold (p < 0.05) at 1 × 10^− 4, 1 × 10^− 5, 1 × 10^− 7 and 1 × 10^− 8 mol/L, respectively. It is noteworthy that ICA at the optimum concentration of 1 × 10^− 6 mol/L could increase the proliferation of hAMSCs from 0.524 ± 0.033-fold to 0.807 ± 0.080-fold (p < 0.05) compared with the control group (Fig. 2).

Effect of different concentrations of ICA on the osteogenic differentiation in hAMSCs

To determine the effect of different concentrations of ICA during hAMSC osteogenic differentiation, the cell morphology at different stages of 1 d, 7 d and 21 d were photographed for morphological observation. As shown in Fig. 3, the cells in each group basically showed a long spindle shape on day 1. Following a 7-day induction period, the morphology of hAMSCs in classic and ICA groups changed from long spindle to polygonal and triangular (Fig. 3). 21 days post-osteoinduction, hAMSCs in both the classic group and ICA group indicided morphological changes: the cells transitioned from the typical long-spindle morphology to short-spindle, triangle, or polygonal shapes with clear boundaries between the cells (Fig. 3).

The marker of bone differentiation is that the osteogenic differentiation of MSC leads to mineralization or the formation of osteoblast nodules. Thus, on day 21 of the osteogenic induction experiment, the formation of mineralized nodules in hAMSCs was evaluated by Alizarin Red Staining (ARS) and the amount of mineralization was quantified by eluting ARS staining from differentiated osteoblasts. As shown in Fig. 4, the treatment in blank group had no nodules formed to minimal ARS staining, indicating that the cells in the blank group did not show mineralization. The cells in the classic group exhibited characteristic osteoblast nodules (Fig. 4). In addition, numerous rose-red calcified nodules in ARS staining were observed in the ICA-1, ICA-2 and ICA-3 groups compared with the blank group, and the most ARS staining was found in ICA-3 group (Fig. 4). This was confirmed by quantitative analysis of ARS, as shown in Table S1.

Effect of different concentrations of ICA on ALP activity of hAMSCs

ALP activity usually reflects the early osteogenic differentiation of hAMSCs. The investigation of ALP activity was done after hAMSCs had been cultured for 1, 3, 7, 14, and 21 days. The ALP activity was not significantly different compare with the blank group, and the cells did not show obvious differentiation on the 1 d and 3 d of induction (Fig. 5). From 7 to 21 days of cultivation, the activity of ALP rose and presented a significantly higher level in the classic group and ICA group than in the blank group. After 7 days of cultivation, compared to the blank group (5.614 ± 0.377), the ALP activity of hAMSCs in ICA-1, ICA-2, ICA-3 and classic group reached 17.491 ± 0.509 (P < 0.01), 43.073 ± 0.422 (P < 0.01), 45.534 ± 1.072 (P < 0.05) and 38.598 ± 0.6444 (P < 0.01). ALP activity in the ICA-1, ICA-2, ICA-3 group (41.656 ± 0.375, 92.222 ± 0.661, 88.195 ± 1.908) and the classic group (63.583 ± 0.615) were significantly higher than that in the blank group (7.457 ± 0.377) on the 14th day. Following a 21-day culture period, the ALP activity of hAMSCs in ICA-1, ICA-2, ICA-3 and classic group reached 92.819 ± 0.509 (P < 0.01), 139.955 ± 1.428 (P < 0.01), 145.102 ± 2.384 (P < 0.01) and 122.056 ± 1.676 (P < 0.01) compared to the blank group (11.175 ± 0.799) (Fig. 5).

Osteogenic inducers are very important for effective stem cell-based treatment of bone defects/losses. Recently, many researches showed that traditional Chinese medicines could induce directional MSC differentiation into osteoblasts [21–25]. These traditional Chinese medicines such as Salvia miltiorrhiza Bunge, Angelica sinensis, Astragalus membranaceus Bunge, Puerarin radix, and Epimedium spp. may be used to promote osteogenesis and inhibit bone resorption, showing that they had a positive effect on the treatment of bone defects/losses [9, 26]. Icariin (ICA) is the natural main active product of Epimedium pubescens, and induces osteogenic differentiation in MSCs by enhancing expression of the osteoblast marker gene runt-related transcription factor 2 (RUNX2) and the bone morphogenetic protein (BMP) [10]. In the present study, our results verified that the potent osteogenic effect on hAMSCs was induced by icariin. The molecular mechanism that ICA induces hAMSCs differentiation into osteogenesis may be that ICA exerts its potent osteogenic effect through the induction of RUNX2 expression, production of BMP, and activation of BMP signaling [9]. Therefore, ICA will be hotspot for bone regenerative medicine due to the extremely low-cost compound and its high abundance.

Although ICA occupied multiple advantages on the proliferation and osteogenic differentiation for bone disease, they might be unexpectedly cytotoxic against stem cells due to a dose-response relationship. In this study, the stimulation effect of ICA on the proliferation of hAMSCs during 3 days of culture was evaluated at various concentrations with 1 × 10^− 4 mol/L, 1 × 10^− 5 mol/L, 1 × 10^− 6 mol/L, 1 × 10^− 7 mol/L and 1 × 10^− 8 mol/L. MTT assay indicated that the hAMSCs pretreated with ICA exhibited increased proliferation comparing with the control group, and the proliferation fold of hAMSCs decreases with the increase of ICA concentration (Fig. 4). Previous studies showed that 0.1–10 µM icariin stimulated the proliferation of rat bone marrow stromal cells (rMSCs), increased the alkaline phosphatase activity, osteoalcin secretion and calcium deposition level of rMSCs during osteogenic induction [27]. Therefore, 1 × 10^− 6 mol/L ICA showed the highest cell viability and osteogenic activity on hAMSCs in the present study, indicating that osteogenic hAMSC induction is sensitive to ICA.

Alkaline phosphatase (ALP) is one of the biochemical markers for osteoblast activity. It is an enzyme in which osteoblasts secrete into the extracellular matrix [28]. ALP can catalyze the hydrolysis of the phosphate esters in an alkaline environment, resulting in the formation of an inorganic phosphate that plays an important role in bone mineralization [29]. Besides, at the processes of the extracellular matrix through the osteoblast, calcium is one of the components and the investigation of calcium deposition becomes remarkable as an indicator of in vitro activities of the osteoblast [30]. Alizarin red staining (ARS), as a marker of calcium deposition in this processes, has been used many related studies to assessing mineralized matrix deposition for osteoblast [9, 30, 31]. In this study, the activity of ALP rose and presented a significantly higher level of activity in the classic group and ICA groups with different concentrations of ICA than in the blank group from 7 to 21 days of hAMSCs cultivation (Fig. 7). At the same time, numerous rose-red calcified nodules in ARS staining were observed in the classic group and ICA groups (Fig. 4). Our study confirmed that ICA would promote hAMSCs differenciation into osteoblast by ALP activity and ARS staining together.

Bone defects/ losses is a serious problem in orthopedics and osteogenic inducers play central roles in effective stem cell-based treatment during the processes. In this study, we have demonstrated that icariin is a safe, effective, and novel natural osteogenic inducer for hAMSCs. When the concentration of ICA was 1 × 10^− 6 mol/L, it is most significant to increase proliferation and promote the osteogenic differentiation of hAMSCs. Therefore, an appropriate amount of icariin might be used as a potential candidate compound for stem cell-based therapy of bone disease.

ALP: Alkaline phosphatase; ARS:Alizarin red staining; DMEM:Dulbecco’s modified eagle medium; DMSO:Dimethyl sulfoxide; FBS:Fetal bovine serum; hAMSCs:human amnion-derived mesenchymal stem cells; ICA:Icariin; MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BMP:Bone morphogenetic proteins; VEGF:Vascular endothelial growth factor; BMSCs:Bone marrow-derived mesenchymal stem cells; MSCs:Mesenchymal stem cells; TE&RM:Tissue engineering and regenerative medicine; AM:Amniotic membrane; PBS:Phosphate-buffered saline; rMSCs:Rat bone marrow stromal cells

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This project was approved by the medical ethics committee of Zunyi Medical University (Approval No. 2018. 246).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by the project of Guizhou science and technology department, grant No. QKHJZ-LKZ[2013]29.g

Authors’ contributions

FW and ZYY designed the study, analyzed the experiments, and wrote the paper. GSQ, WH, KW and YLZ carried out the data collection and data analysis and revised the paper. All authors read and approved the final version of the manuscript.

Acknowledgements

All authors would like to thank to the key laboratory of basic pharmacology of ministry of education and the joint international research laboratory of ethnomedicine of ministry of education.

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Effects of icariin on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells

Status:

Journal Publication

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials And Methods

Results

Morphology and phenotype of hAMSCs

Effect of different concentrations of ICA on the proliferation of hAMSCs

Effect of different concentrations of ICA on the osteogenic differentiation in hAMSCs

Effect of different concentrations of ICA on ALP activity of hAMSCs

Discussion

Conclusions

Abbreviations

Declarations

Availability of data and materials

Ethics approval and consent to participate

Funding

Authors’ contributions

Acknowledgements

References

Supplementary Files

Status:

Journal Publication

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