Kim, Ji-Hun
(College of Veterinary Medicine, Kyungpook National University)
,
Yun, Sungho
(College of Veterinary Medicine, Kyungpook National University)
,
Seo, Min-Su
(Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation)
,
Bae, Seulgi
(College of Veterinary Medicine, Kyungpook National University)
,
Jang, Min
(College of Veterinary Medicine, Kyungpook National University)
,
Ku, Sae-Kwang
(Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University)
,
Kwon, Young-Sam
(College of Veterinary Medicine, Kyungpook National University)
,
Lee, Hae Beom
(College of Veterinary Medicine, Chungnam National University)
The purpose of this study is to investigate the clinical effects of carboxymethyl chitosan (CMC) and adipose-derived mesenchymal stem cells (MSCs) on osteoarthritis (OA). Thirty New Zealand white rabbits were used as cranial cruciate ligament transection and partial meniscectomy models. The rabbits ...
The purpose of this study is to investigate the clinical effects of carboxymethyl chitosan (CMC) and adipose-derived mesenchymal stem cells (MSCs) on osteoarthritis (OA). Thirty New Zealand white rabbits were used as cranial cruciate ligament transection and partial meniscectomy models. The rabbits were divided into five groups (n = 6) according to the intra-articular injection materials: the control group with PBS, the HA group with hyaluronic acid, the CMC group with CMC, the MSC group with MSCs emerged in PBS, and the MSC+CMC group with CMC and MSCs. Knee thickness, extension angle, gross morphology, histopathology and immunohistochemistry were performed to evaluate the effects of CMC and MSCs on rabbit OA. On the morphologic and histologic examination, the articular surfaces of the femur and tibia were markedly damaged in control group with higher Mankin score and lower cartilage surface thickness. However, OA related cartilage defects were alleviated by the treatment of MSC and/or CMC. The expressions of apoptotic and inflammatory cytokines were decreased and cartilage extracellular matrix (ECM) related collagens I and II were enhanced by the treatment of MSC and/or CMC. In conclusion, this study showed that CMC and MSC treatments have a beneficial effects on OA via the protection of cartilage damage, the stimulation of ECM, and the inhibition of inflammatory and apoptotic reaction.
The purpose of this study is to investigate the clinical effects of carboxymethyl chitosan (CMC) and adipose-derived mesenchymal stem cells (MSCs) on osteoarthritis (OA). Thirty New Zealand white rabbits were used as cranial cruciate ligament transection and partial meniscectomy models. The rabbits were divided into five groups (n = 6) according to the intra-articular injection materials: the control group with PBS, the HA group with hyaluronic acid, the CMC group with CMC, the MSC group with MSCs emerged in PBS, and the MSC+CMC group with CMC and MSCs. Knee thickness, extension angle, gross morphology, histopathology and immunohistochemistry were performed to evaluate the effects of CMC and MSCs on rabbit OA. On the morphologic and histologic examination, the articular surfaces of the femur and tibia were markedly damaged in control group with higher Mankin score and lower cartilage surface thickness. However, OA related cartilage defects were alleviated by the treatment of MSC and/or CMC. The expressions of apoptotic and inflammatory cytokines were decreased and cartilage extracellular matrix (ECM) related collagens I and II were enhanced by the treatment of MSC and/or CMC. In conclusion, this study showed that CMC and MSC treatments have a beneficial effects on OA via the protection of cartilage damage, the stimulation of ECM, and the inhibition of inflammatory and apoptotic reaction.
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가설 설정
It was hypothesized that intra-articular injection of MSCs suspended in CMC could be protective and regenerative effect on OA as minimal invasive treatment. The purpose of this study is to evaluate the effect of CMC alone or in combination with MSC on surgically-induced OA in rabbits using gross morphologic, histologic and immunohistochemical examinations, and to compare the results with those of HA, and consequently to establish new therapeutic regime.
제안 방법
Each femur and tibia articular surface cartilages was longitudinally trimmed, and then embedded in paraffin, sectioned(3-4 µm) using tungsten blade equipped automated polycut microtome (Model RM2255, Leica, Wetzlar, Germany) and stained with Sirius red stain for cartilaginous tissues. In each prepared histological samples, the histological profiles were interpreted under a light microscope (Model Eclipse 80i, Nikon, Tokyo, Japan)as blinds to group distribution when this analysis was made.
The immunoreactivities of caspase-3 and PARP for detection of the cell apoptosis by OA was examined to observe cell protective effects of MSC and/or CMC on the articular cartilages in this study. The decrease of cartilage ECM was associated with death or apoptosis of chondrocyte (5).
The intra-articular injections of treatment materials were performed per week, total 3 times from 5 weeks of OA modeling to 7 weeks according to the groups: the sham group without treatment, the control group with 0.3 ml of PBS, the HA group with 0.3 ml of hyaluronic acid, the CMC group with 0.3 ml of CMC, the MSC group with 2.0 × 106 MSCs in 0.3 ml of PBS, and the MSC+CMC group with 2.0 × 106 MSCs in 0.3 ml of CMC.
It was hypothesized that intra-articular injection of MSCs suspended in CMC could be protective and regenerative effect on OA as minimal invasive treatment. The purpose of this study is to evaluate the effect of CMC alone or in combination with MSC on surgically-induced OA in rabbits using gross morphologic, histologic and immunohistochemical examinations, and to compare the results with those of HA, and consequently to establish new therapeutic regime.
The stifle thickness was measured with caliper on transverse direction at the level of thickest part of the joint at 0 day, 5 weeks, 6 weeks, 7 weeks, and 8 weeks. One veterinarian measured the stifle diameter to reduce a variation.
대상 데이터
In this study, the CD34 positive cells, which is regarded as hematopoietic progenitor cells (43), were detected on the articular surface in MSC and/or CMC treated rabbits. As MSCs do not express CD34, further study should be necessary to find out whether these CD34 positive cells are the results of the proliferation of resident cells or migration of cells with unidentified origin or phenotype alteration of injected MSCs.
Thirty New Zealand white rabbits were used (Saeronbio Inc., Gyeonggi, Korea). The body weights of rabbits were 2.
데이터처리
A multiple comparison tests for different groups were conducted. After the variance homogeneity test with the Levene test, the obtain data were analyzed with one way ANOVA test followed by least-significant differences multi-comparison (LSD) test for parametric analysis, and Kruskal-Wallis H test was conducted followed by the Mann-Whitney U (MW) test for nonparametric comparison test. Statistical analyses were conducted using SPSS software (version 23, IBM SPSS Inc.
성능/효과
From this aspect, we performed immunostaining for the caspase-3 and PARP expression whether apoptotic change during OA could be affected by MSC and/or CMC treatment. In this study, we found that the number of caspase-3 and PARP positive cells increased in the OA condition and decreased with a treatment of MSC and/or CMC. These findings are consistent with the results of previous study that caspase-3 led to PARP to cleavage and consequently caused the progression of OA with cell apoptosis and death (33).
MSCs were collected and used between passage and 2 in all the experiments of this paper. The flow cytometry analysis with established MSCs was performed, and obtained MSCs were negative for cluster of differentiation (CD) 34 and CD 45 and strongly positive for CD 29 and CD 44.
These results is considered that CMC, MSC or their combination treatment favorably ameliorates the surgically-induced OA in rabbits via ECM synthesis and via anti-apoptotic and anti-inflammatory effects. Therefore, the combination of CMC and MSC may be useful as a novel strategy for the treatment of OA.
후속연구
In this study, the CD34 positive cells, which is regarded as hematopoietic progenitor cells (43), were detected on the articular surface in MSC and/or CMC treated rabbits. As MSCs do not express CD34, further study should be necessary to find out whether these CD34 positive cells are the results of the proliferation of resident cells or migration of cells with unidentified origin or phenotype alteration of injected MSCs.
In addition, it has been reported that chitosan had an antiinflammatory effect on the lipopolysaccharide induced in vitro inflammation model (42). The evidence of synergistic effect of MSC and CMC was noted in this study, however further study would be necessary for precise mechanism of interaction.
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