Effects of low-intensity pulsed ultrasound on the proliferation of human umbilical cord mesenchymal stem cells and their chondrogenic differentiation via BMP2 /smad signaling pathway

Background: Although Low-intensity pulsed ultrasound (LIPUS) regiment has been applied to the treatment of cartilage injury clinically, the effect of LIPUS stress loading on the proliferation and differentiation of hUCMSCs is still controversial. Yet the specific mechanical mechanism of LIPUS stimulates cartilage differentiation of hUCMSCs has not been clarified. Objective ： To investigate the effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) via BMP2 /smad signaling pathway in vitro. with other groups, Alcian blue staining of GAG , COL II fluorescent staining in the LIPUS group were much stronger, quantitative RT-PCR assays showed that the expression of COL II, GAG, Sox-9, smad1, smad5 and smad9 in LIPUS group was significantly increased. Conclusion ： The suitable LIPUS irradiation can promote cell proliferation, 50mW/cm 2 intensity at 5min/d is most significant. And it can promote chondrogenesis of hUCMSCs via upregulating MP2 /smads signaling pathway. analysis of variance (ANOVA) and two groups by pair-wise Student’s t test. P values < 0.05 were considered statistically significant.


Introduction
Stem cell based tissue engineering has emerged as a promising strategy for articular cartilage regeneration [1][2][3][4]. Human umbilical cord mesenchymal stem cells (hUCMSCs) derived from Wharton jelly tissues has the characteristics of stem cells and pre-chondrocytes, and naturally expresses Sox-9 and COL II positively, and it has the ability to differentiate and express multi-directional cells stably under specific conditions and stably express specific cell phenotypes. These characteristics make hUCMSCs an ideal seed cell for cartilage tissue engineering [5][6][7][8].
It is well known that stress loading is one of the most basic biological stimulation to cells in the process of biological evolution. MSCs can recruit, proliferate and 4 differentiate into chondroblast under stress stimulation, which is the cytological basis of new cartilage formation [9][10][11][12][13]. In recent years, it has been found that Low intensity pulsed ultrasound intensity ultrasound (LIPUS) integrates mechanical stimulation of compressive stress and fluid shear stress through high frequency and small amplitude pulsed pressure wave. LIPUS direct act on joints can improve the nutrition of articular cartilage, reduce the production of inflammatory mediators, promote the repair of articular cartilage defects and promote the proliferation, directional differentiation and homing of stem cells in vitro and in Vivo. It has been gradually applied to the repair and healing treatment of cartilage injury clinically [14][15].
However, the effect of LIPUS stress loading on the proliferation and differentiation of hUCMSCs is controversial [16][17][18][19][20].The suitable parameters of LIPUS including irradiation intensity and time need to be clarified. The specific mechanical mechanism of LIPUS stimulates cartilage differentiation and proliferation of hUCMSCs has not been fully elucidated. Therefore, in our study, by evaluating the proliferation of hUCMSCs under different parameters of LIPUS loading conditions, suitable LIPUS loading conditions that could produce good chondrogenic differentiation effect on hUCMSCs in vitro were selected, and then the suitable parameter of LIPUS was loaded into hUCMSCs for chondrogenic differentiation and its effects through the BMP2 /Smad signaling 5 pathway was further evaluated. Biotechnology, Guangzhou,China) with 10% fetal bovine serum (Saiye Biotechnology, Guangzhou,China),100mg/mL streptomycin, and 100U/mL penicillin at 37℃ in an incubator with 95% air and 5% CO2. After 24h, the non-adherent cells were discarded by fluid exchange, and the fluid was changed every 2-3 days. When the cell fusion reached 80%, the cells were digested by trypsin and passaged at the ratio of 1:2.

Materials and methods
The cells were characterized morphologically under an inverted microscope (Olympus, Tokyo, Japan). Phenotype of hUCMSCs including CD29, CD34, CD44, 6 CD45, CD73and CD105 was identified by flow cytometry, CD34 and CD45 are markers of haematopoietic stem cell.

CCK-8 assay of cell viability
P4 of hUCMSCs growing on 96-well plates at a density of 100 uL with 1.5 ×10 4 /ml were randomly divided into four groups: control group, L30 group (30 mW/cm 2 ), L50 group (50 mW/cm 2 ), L80 group (80 mW/cm 2 ). The LIPUS irradiation groups exposed the cell culture plate to the probe element of the SonaCell device through the coupling agent and placed it in the cell incubator for LIPUS stimulation at 5min/d, 10min/d or 20 min/d respectively for 5 consecutive days, the control group was treated without LIPUS. And the culture medium was replaced after 24h .Another blank hole was taken and added into the culture medium as blank group. 10 μl of CCK-8 solution was added 24h, 48h, 72h, 96h and 120h later. Cells were cultured for an additional 4 hours and the absorbance at 450nm was measured using a microplate reader. The proliferation inhibition rate = (OD of experimental group -OD of blank group)/(OD of control group -OD of blank group)x100%. Four groups of repeated experiments were performed.The data were statistically analyzed.

Alcian blue staining
After the hUCMSCs were cultured in chondrogenic medium for 3 weeks, the cells of each group were washed with PBS three times, fixed with 4% paraformaldehyde at room temperature for 30min, PBS washed 3 times, 0.1M HCl solution was adde for 5 minutes, pH value drops to 1.0; 1% alein blue staining for 30 minutes; PBS washed thrice.Then the cells were observed under an inverted microscope, and took pictures with Image-ProPlus software.

Immunofluorescence staining
After the hUCMSCs were cultured in chondrogenic medium for 3 weeks, and the cells were washed thrice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and then washed with PBS three tims, treated with 0.5%  Table 1.

Statistical analysis
All data were expressed as mean ± standard deviation (SD) of three independent experiments and analyzed using SPSS 22.0 software (IBM Corp, Armonk, NY, USA).
Multiple groups were compared by single-factor analysis of variance (ANOVA) and two groups by pair-wise Student's t test. P values < 0.05 were considered statistically significant. 10

The effect of LIPUS irradiation on cell proliferation
The results of hUCMSCs proliferation of each group after irradiating with different LIPUS intensities and duration time are shown (Fig. 1). At 5min/d, the cell proliferation rate of the L50 group was the highest (P<0.05). But the cell proliferation ability decreased with the increase of irradiation intensity and the pulse duration instead, cell proliferation is weakest at L80 group (80w/cm 2 , 20min/d) (P<0.05) .   We observed that the expression of COL II, GAG and Sox9, and the expression of BMP2, Smad1, Smad5 and Smad9 in the Noggin group were significantly lower than that of the LIPUS group, LIPUS+Noggin group. However, compared with the control 16 group, the expression of smad1 and smad9 increased rather than decreased, while the expression of smad5 was no significant difference. Is it possible that the addition of the Noggin, an exogenous BMP2 inhibitor, may not completely inhibit the activation of BMP2? More work needs to be done to further explored. In addition, we found that the expression of Sox-9, COL II, and GAG in LIPUS+Noggin group was higher than the control group. Whether this may be related to the activation of other signaling pathways in the process of LIPUS promoting cartilage differentiation. More studies have verified that the interaction between multiple signaling pathways in the process of chondrogenic differentiation [24,25].
In conclusion, LIPUS irradiation with suitable parameters can promotes the proliferation and chondrogenesis of hUMSCs, and the BMP2 /smads signaling pathway plays an important positive regulatory role in the process. Our findings uncover the mechanism of the effects of LIPUS on hUMSCs and provide some promising treatment for articular cartilage defects in the future.

Ethics approval and consent to participate：
The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of