This study investigated the efficacy of transplantation of allogeneic adipose-derived stem cells (ADMSCs) in rats with knee osteoarthritis (KOA) and the effect on the expression of matrix metalloproteinase 13 (MMP-13) and discoid domain receptor 2 (DDR2). In total, sixty rats were randomly selected. Eleven rats were selected as the blank group. Forty-four rat KOA models were established, and the remaining 5 rats were used for stem cell extraction. The rats were randomly divided into two groups, and the transplantation group was treated with ADMSCs transplantation. The KOA group was intragastrically administered with saline. The expressions of MMP-13 mRNA and DDR2 in rats were detected by RT-qPCR and immunohistochemistry. Correlation analysis was performed in MMP-13 mRNA and DDR2 expression levels in the KOA rats. After treatment, the indexes of Lequesne MG knee joints, MMP-13 mRNA and DDR2 in the transplanted rats were significantly lower than those in the KOA group (P<0.05). In the KOA rats, MMP-13 mRNA and DDR2 was positively correlated (r=0.830, P<0.001). Therefore, the transplantation of ADMSCs has a significant effect on the KOA rats, which can effectively improve the knee joint function of KOA rats and reduce the expression of MMP-13 mRNA and DDR2 in rats, and it is worthy of clinical promotion.
Knee osteoarthritis (KOA) is a degenerative disease that occurs in the elderly and has a high incidence. It is one of the main causes of activity disorder and joint pain in the elderly, threatening life and health as well as the quality of life for the elderly (
Matrix metalloproteinase-13 (MMP-13), a key enzyme that mediates the degradation of extracellular matrix, can degrade 90% of type II collagen in the extracellular matrix (
However, the current research on the treatment of KOA with adipose-derived stem cell transplantation is rare. Therefore, the effects of allogeneic adipose-derived stem cell (ADMSCs) transplantation on the KOA rats and the effects on MMP-3 and DDR2 were investigated, in order to find a better treatment for KOA patients.
Sixty healthy female SD rats aged 8 weeks old and weighing 171.61±7.52 g were selected. All rats were purchased from Shanghai Slack Laboratory Animals Co., Ltd. [(Shanghai, China), production license SCXK (Shanghai) 2012-0002], The rats were maintained in plastic boxes with solid padding at a constant temperature of 22°C, relative humidity (50–65%), in a 12:12-h light/dark cycle, and were allowed free access to food and water. The operation of the modeling process on rats was in accordance with the standards of the Experimental Animal Ethics Committee of Shanghai East Hospital Tongji University (Shanghai, China). Type II collagenase was purchased from Sigma; Merck KGaA (Darmstadt, Germany); rabbit anti-mouse DDR2 polyclonal antibody was purchased from ImmunoWay Biotechnology Company (Plano, TX, USA); TRIzol reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA); PCR fluorescence quantitative kit and reverse transcription kit were purchased from Dalian Takara Biotechnology Co., Ltd. (Dalian, China).
Eleven rats were randomly selected as the blank group and continued to be kept routinely without any intervention. The rat KOA model was established by collagenase injection (
After anesthetizing 5 rats, a part of the subcutaneous adipose tissue was taken out from the groin of the rat and placed in a petri dish, and connective tissue and large blood vessels in the tissue were excised with ophthalmic scissors and the tissue was washed with PBS to a bloodless color. After washing, the tissue was cut and placed in a centrifuge tube; 0.1% type I collagenase was added to the centrifuge tube and mixed uniformly, and then subjected to water bath digestion at 37°C. After digestion, centrifuged at 400 × g for 5 min at 4°C, the cells were then resuspended in DMEM complete medium containing 10% FBS. Then the cells were inoculated in culture flasks, the cell density was adjusted to 5×410/cm2 and culture was continued in a 37°C, 5% CO2 incubator. When the cells were adherently grown to 80%, the cells were harvested for subsequent stem cell transplantation.
Venous blood (2 ml) was taken from the rats before and after treatment, and total MMP-13 mRNA in the blood was extracted by adding TRIzol reagent. The concentration and quality of total RNA were detected by ultraviolet spectrophotometer (purchased from ShanghaiMetash Instruments Co., Ltd., Shanghai, China). Total RNA (2 µl) was taken and cDNA was synthesized in strict accordance with the instructions of the reverse transcription kit. The synthesized cDNA (2 µl) was subjected to SYBR-Green PCR kit (Beyotime, Shanghai, China), and the reaction conditions of MMP-13 were: 95°C for 4 min, then 95°C for 45 sec, 60°C for 30 sec, 72°C for 30 sec, and 40 cycles. The expression level of MMP-13 mRNA was detected by using β-actin as an internal reference substance. The primer sequences are shown in
The rats were sacrificed and the knee joint tissue samples were taken. The samples were fixed in 4% paraformaldehyde buffer for 24 h at 4°C, and made paraffin blocks after taken out, then embedded in sections with a thickness of 3 µm, and then blocked with 5% milk at room temperature for 1.5 h. A total of 50 µl of primary anti-DDR2 monoclonal antibody (1:500; cat. no. kl974Hu21; Shanghai Kanglang Biotechnology Co., Ltd., Shanghai, China) was added dropwise, and the mixture was allowed to stand overnight at 4°C. Then, the sample was rinsed with PBS 3 times for 5 min each time. A total of 50 µl of secondary rabbit anti-mouse monoclonal antibody (1:500; cat. no. PAB9358; Shanghai Qunji Biotech Co., Ltd., Shanghai, China) was added, and let stand in a 37°C incubator for 20 min, then rinsed with PBS 3 times, then streptavidin-peroxidation enzyme was added and further allowed to stand at 37°C for 20 min, rinsed with PBS 3 times, and then added with DAB for color development (20:1:1). When microscopic observation showed appropriate staining degree, the sample was rinsed with tap water for 15–20 min to terminate the reaction, followed by hematoxylin repeated dyeing for 2–3 min, rinsed again with tap water for 15–20 min. Finally, conventional dehydration and sealing were performed.
When brown or yellow-brown particles appeared in the cytoplasm, DDR2 was positively expressed, and 10 fields were randomly selected for positive cell count.
i) Three groups of rats were evaluated for Lequesne MG knee joint evaluation level before intervention treatment and 6 weeks after intervention treatment (
In this study, the SPSS 18.0 software package [Bo Yi (Beijing) Information Technology Co., Ltd., Beijing, China] was used for statistical analysis of the experimental data. The measurement data were expressed using mean ± standard deviation. The two groups were compared by t-test. Multiple groups were compared using analysis of variance. LSD/t test was used for post hoc comparison. Paired t-test was used for comparison before and after treatment within the group. Pearson's correlation analysis was used for correlation. The experimental pictures were drawn using GraphPad Prism 6 software (Emerald Biotech Co,. Ltd., Hangzhou, China). P<0.05 for the difference was statistically significant.
The Lequesne MG knee joint evaluation indexes of the transplantation and KOA groups were significantly higher than those of the blank group, and the difference was statistically significant (P<0.05), but there were no significant differences in the indexes of the Lequesne MG knee joint evaluation between the transplantation and KOA groups (P>0.05). After the treatment, the indexes of Lequesne MG knee joints in the transplantation group were lower than those in the KOA group, and the difference was statistically significant (P<0.05). There were no significant differences in the indexes of the rats in comparison to the blank group (P>0.05) (
There was no significant difference in the expression of MMP-13 mRNA between the transplanted and KOA rats before treatment (P>0.05), but they were significantly higher than those in the blank group, and the difference was statistically significant (P<0.05); MMP-13 mRNA expression in the transplantation group was significantly lower than that in the KOA group, and the difference was statistically significant (P<0.05). Although the expression level of MMP-13 mRNA in the transplantation group after treatment was still higher than that of the blank group, there was no significant difference (P>0.05) (
There was no significant difference in the expression of DDR2 between the transplanted and KOA rats before treatment (P>0.05), but they were significantly higher than those in the blank group, and the difference was statistically significant (P<0.05); DDR2 positive expression in the transplantation group was significantly lower than that in the KOA group after treatment, and the difference was statistically significant (P<0.05). Although the positive expression level of DDR2 in the transplantation group after treatment was still higher than that of the blank group, there was no significant difference (P>0.05) (
According to Pearson's correlation analysis, there was a positive correlation between MMP-13 mRNA and DDR2 in KOA rats (r=0.830, P<0.001), as shown in
As a chronic degenerative bone and joint disease, in recent years, with the aging of the population, the incidence of KOA is increasing, and the incidence of KOA in the elderly aged over 65 years is even as high as 50% or more, causing a heavy burden to the family (
After establishing the model, rats were divided into groups and transplanted with ADMSCs. The results showed that the indexes of Lequesne MG knee joints in the transplantation group were lower than those in the KOA group, and the difference was statistically significant (P<0.05), which indicates that ADMSCs transplantation therapy has a significant effect on improving knee joint score in rats. Some studies (
MMP-13 is an MMP that can effectively degrade various collagens. Studies have reported (
In summary, the transplantation of ADMSCs has a significant effect on KOA rats, which can effectively improve the knee joint evaluation of KOA rats as well as the expression of MMP-13 mRNA and DDR2 in rats, and promote the recovery of rats, which is worthy of clinical promotion. However, more in-depth experiments are still required.
Not applicable.
This study was supported by the Scientific Research Project of Shanghai Science and Technology Committee (17411968400).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JL drafted the manuscript. JL and XZ contributed to the isolation and culture of adipose stem cells. QS and FX were responsible for the rat KOA model construction. JL and GS performed PCR. All authors read and approved the final manuscript.
The study was approved by the Experimental Animal Ethics Committee of Shanghai East Hospital Tongji University (Shanghai, China).
Not applicable.
The authors declare that they have no competing interests.
Correlation analysis between MMP-13 mRNA and DDR2 in KOA rats. There was a positive correlation between MMP-13 mRNA and DDR2 in KOA rats (r=0.830, P<0.001). MMP-13, matrix metalloproteinase 13; DDR2, discoid domain receptor 2; KOA, knee osteoarthritis.
Related primer sequences.
Factors | Forward primer sequences | Reverse primer sequences |
---|---|---|
MMP-13 | 5′-TGACTATGCGTGGCTGGA-3′ | 5′-GGGAAGGGGCTAATGAACA-3′ |
β-actin | 5′-CACACTGTGCCCATCTACGA-3′ | 5′-CTCAGTGAGGATCTTCATGAGGTAGT-3′ |
MMP-13, matrix metalloproteinase 13.
Evaluation of Lequesne MG knee joint before treatment in the three groups of rats.
Indexes | Transplantation group (n=20) | KOA group (n=20) | Blank group (n=11) | F value | P-value |
---|---|---|---|---|---|
Pain stimulation | 3.71±0.25 |
3.69±0.23 |
1.21±0.13 | 543.6 | <0.001 |
Gait | 3.81±0.18 |
3.75±0.21 |
1.07±0.05 | 1,029.0 | <0.001 |
Joint swelling | 2.95±0.23 |
2.93±0.22 |
1.08±0.04 | 369.2 | <0.001 |
Joint activity | 3.67±0.19 |
3.69±0.18 |
1.12±0.09 | 981.6 | <0.001 |
P<0.05, compared with the blank group. KOA, knee osteoarthritis.
Evaluation of Lequesne MG knee joint after treatment in the three groups of rats.
Indexes | Transplantation group (n=20) | KOA group (n=20) | Blank group (n=11) | F value | P-value |
---|---|---|---|---|---|
Pain stimulation | 1.14±0.15 |
3.65±0.11 | 1.11±0.10 |
891 | <0.001 |
Gait | 1.05±0.04 |
3.69±0.19 | 1.07±0.06 |
2,689.0 | <0.001 |
Joint swelling | 1.08±0.03 |
3.01±0.25 | 1.03±0.02 |
916.2 | <0.001 |
Joint activity | 1.12±0.09 |
3.71±0.14 | 1.09±0.06 |
3,510 | <0.001 |
P<0.05, compared with the KOA group. KOA, knee osteoarthritis.
MMP-13 mRNA expression levels before and after treatment in the three groups of rats.
Time | Transplantation group (n=20) | KOA group (n=20) | Blank group (n=11) | F value | P-value |
---|---|---|---|---|---|
Before treatment | 2.27±0.58 | 2.19±0.51 | 1.02±0.22 | 25.78 | <0.001 |
After treatment | 1.13±0.26 | 2.38±0.49 | 1.04±0.24 | 74.87 | <0.001 |
t value | 8.021 | 1.201 | 0.204 | – | – |
P-value | <0.001 | 0.237 | 0.841 | – | – |
MMP-13, matrix metalloproteinase 13; KOA, knee osteoarthritis.
DDR2 positive expression levels before and after treatment in the three groups of rats (%).
Time | Transplantation group (n=20) | KOA group (n=20) | Blank group (n=11) | F value | P-value |
---|---|---|---|---|---|
Before treatment | 28.61±3.66 | 28.59±3.57 | 18.21±2.35 | 40.50 | <0.001 |
After treatment | 18.72±2.51 | 28.21±3.52 | 17.92±2.29 | 68.65 | <0.001 |
t value | 9.966 | 0.339 | 0.293 | – | – |
P-value | <0.001 | 0.737 | 0.772 | – | – |
DDR2, discoid domain receptor 2. KOA, knee osteoarthritis.