LncRNA NONHSAT030515 promotes the chondrogenic differentiation of human adipose-derived stem cells via regulating the miR-490-5p/BMPR2 axis

Abstract Background Chondrogenic differentiation of human adipose-derived stem cells (hADSCs) is important for cartilage generation and degradation. LncRNAs play an essential role in stem cell differentiation. However, the role and mechanism of lncRNA in hADSCs remain unclear. Our previous study showed that miR-490-5p was downregulated during chondrogenic differentiation of hADSCs. In this study, we investigated the effect and mechanism of lncRNA NONHSAT030515 interacting with miR-490-5p on chondrogenic differentiation of hADSCs. Methods Alcian blue staining was used to assess the deposition of chondromatrix proteins following chondrogenic differentiation of human adipose stem cells. Immunohistochemistry was used to evaluate the expression of collagenII. TargetScan, miRTarBase and miRDB database analyses were applied to find the miRNA and target genes of lncRNA NONHSAT030515. A dual luciferase experiment was conducted to identify the direct target of NONHSAT030515. pcDNA3.1- NONHSAT030515 transfection and sh- NONHSAT030515 treatment were conducted to verify the role of lncRNA NONHSAT030515 in chondrogenic differentiation. The levels of Aggrecan, SOX9 and COL2A1 were analyzed by qRT-PCR and Western blot assay. Results Alcian blue staining, immunocytochemical, qRT-PCR, and Western blot have determined that lncRNA NONHSAT030515 can promote the chondrogenic differentiation of hADSCs. MiR-490- 5p was the direct target of NONHSAT030515, while BMPR2 was the target gene. This result was confirmed by luciferase reporter assay. Up-regulation of NONHSAT030515 promoted BMPR2 protein expression and promoted chondrogenic differentiation, whereas down-regulation of NONHSAT030515 caused completely opposite results. Conclusion LncRNA NONHSAT030515 promotes the chondrogenic differentiation of hADSCs through increasing BMPR2 expression by regulating miR-490- 5p.

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The Effect of Human Platelet-Rich Plasma and L-Ascorbic Acid on Morphology, Proliferation, and Chondrogenesis Ability towards Human Adipose-Derived Stem Cells

Molecular and Cellular Biomedical Sciences ◽

10.21705/mcbs.v3i1.43 ◽

2019 ◽

Vol 3 (1) ◽

pp. 26 ◽

Cited By ~ 5

Author(s):

Imam Rosadi ◽

Karina Karina ◽

Iis Rosliana ◽

Siti Sobariah ◽

Irsyah Afini ◽

...

Keyword(s):

Stem Cells ◽

Ascorbic Acid ◽

Alcian Blue ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Cartilage Tissue ◽

Population Doubling ◽

Blue Staining ◽

Alcian Blue Staining ◽

Adipose Derived Stem Cells

Background: Microtia is a congenital malformation in the external ear due to cartilage defect. Adipose-derived stem cells (ADSC) is promising cells to develop cartilage tissue engineering for microtia. In this study, we focused on proliferation and chondrogenesis of ADSC in three different media, which consist of 10% fetal bovine serum (FBS), 10% FBS with L-ascorbic acid, and 10% human platelet rich plasma (PRP). Methods: ADSC were induced to differentiate into adipocytes, chondrocyte and osteocytes. ADSC morphology, proliferation and population doubling time was compared in three different media and analysed. Observation and alcian blue staining were done every 7 days to assess chondrogenic potency of ADSC from each treatment.Results: Isolated ADSC were able to differentiate into adipocytes, osteocytes and chondrocytes. ADSC in all group have fibroblast-like morphology, but cells in 10% FBS and 10% FBS with LAA group were flattened and larger. ADSC in 10% PRP group proliferates faster than 10% FBS with and without LAA. PDT values of ADSC were 34 hours, 44 hours and 48 hours, respectively for 10% PRP, 10% FBS with LAA and 10% FBS group. Alcian blue staining revealed that ADSC in 10% FBS with LAA and 10% PRP were able to proceed to chondrogenesis when cultured time were prolong up to 21 days, but not with ADSC in 10% FBS. Conclusion: We conclude that adding 10% FBS with LAA or 10% PRP into medium culture can support proliferation and chondrogenesis of ADSC. Key words: human ADSC, PRP, L-ascorbic acid, proliferation, chondrogenesis

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miR-134 inhibits chondrogenic differentiation of bone marrow mesenchymal stem cells by targetting SMAD6

Bioscience Reports ◽

10.1042/bsr20180921 ◽

2019 ◽

Vol 39 (1) ◽

Cited By ~ 2

Author(s):

Shaogang Xu ◽

Xuejian Wu

Keyword(s):

Bone Marrow ◽

Alcian Blue ◽

Chondrogenic Differentiation ◽

Luciferase Reporter ◽

Blue Staining ◽

Alcian Blue Staining ◽

Reporter Assay ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells ◽

Dual Luciferase Reporter Assay

Abstract Various miRNAs have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs); however, whether miR-134 plays a role in this biological process remains undetermined. In the present study, we first evaluated the chondrogenic differentiation of BMSCs by Alcian blue staining, and examined the miR-134 expression by quantitative real-time PCR (qRT-PCR) during this process. And miR-134 inhibitor was used to investigate the functions of miR-134 in chondrogenic differentiation of BMSCs by Alcian blue staining, qRT-PCR, and Western blot. Subsequently, the correlation between miR-134 and SMAD6 was assessed via bioinformatics analysis and dual-luciferase reporter assay. Finally, the role of SMAD6 in chondrogenic differentiation of BMSCs was also determined through Alcian blue staining, qRT-PCR, and Western blot. As results showed that miR-134 expression was significantly down-regulated during chondrogenic differentiation, and inhibition of miR-134 obviously promoted chondrogenic differentiation. Dual-luciferase reporter assay indicated that miR-134 could directly target the 3′-UTRs of SMAD6, inhibit miR-134 expression in BMSCs, and up-regulate SMAD6 expression. Moreover, we found that overexpression of SMAD6 significantly promoted chondrogenic differentiation, and that SMAD6-induced promotion of chondrogenic differentiation could be reversed by miR-134 mimics. In conclusion, our findings suggest that miR-134 may act as a negative regulator during chondrogenic differentiation of BMSCs by interacting with SMAD6.

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Silencing Smad7 Potentiates BMP2-induced Chondrogenic Differentiation and Inhibits Endochondral Ossification in Human Synovial Derived Mesenchymal Stem Cells

10.21203/rs.3.rs-96609/v1 ◽

2020 ◽

Author(s):

pengcheng xiao ◽

Zhenglin Zhu ◽

Chengcheng Du ◽

Yongsheng Zeng ◽

junyi Liao ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Chondrogenic Differentiation ◽

Endochondral Ossification ◽

Cartilage Tissue Engineering ◽

Cartilage Tissue ◽

Blue Staining ◽

Alcian Blue Staining ◽

Cartilage Injuries

Abstract Background: Cartilage injuries pose formidable challenges for effective clinical management. Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries. Bone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factors for transgene-enhanced cartilage tissue engineering. However the BMP2 is failed to maintain a stable chondrogenic phenotype as it also induces robust endochondral ossification. Recently, human synovial derived mesenchymal stem cells (hSMSCs) arouse interested through the poor differentiation potential into osteogenic lineage. Smad7, a protein to antagonizes TGF-β/BMP signaling pathway has been discovered significant in the endochondral ossification. In the present study ,we further explore the effect of downregulate Smad7 in BMP2-induced chondrogenic differentiation of hSMSCs. Methods: hSMSCs were isolated from synovium of human knee joint through adhesion growth. In vitro and in vivo chondrogenic differentiation models of hSMSCs were constructed . Transgenes of BMP2, silencing Smad7 and Smad7 were expressed by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining. The chondrogenic differentiation was detected by alcian blue staining. Gene expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), Immunofluorescence and immunohistochemistry. The subcutaneous stem cell implantation model was established and evaluated by micro-CT , h＆e staining, alcian blue staining and immunohistochemistry assay.Results: Compared to other MSCs, hSMSCs performed less of capability to osteogenic differentiation. But the occurrence of endochondral ossification is still inevasible during BMP2 induced cartilage formation. We found that silencing Smad7 enhanced the BMP2-induced chondrogenic differentiation of hSMSCs in vitro. Also, it leading to much less of hypertrophic differentiation. The subcutaneous stem cells implantation assays demonstrated silencing Smad7 potentiates BMP2-induced cartilage formation and inhibits endochondral ossification. Conclusion: This study strongly suggests that application of hSMSCs , cell scaffolds and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibit the expression of Smad7 in BMP2-induced hSMSCs differentiation may be a new strategy for cartilage tissue engineering.

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Effects of experimental periodontitis on proliferation and osteogenic differentiation of adipose-derived stem cells in rats

10.21203/rs.2.18744/v1 ◽

2019 ◽

Author(s):

Yan Li Huang ◽

Le Da Cheng ◽

Ya Jie Fan ◽

Yi Wang ◽

Baoyu Zhu ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Western Blot ◽

Osteogenic Differentiation ◽

Male Rats ◽

Cell Counting ◽

Standard Diet ◽

Adipose Derived Stem Cells ◽

Rt Pcr ◽

Qrt Pcr

Abstract Background and Objective: The application of ASCs in periodontal regeneration is a good choice. Inflammatory micro-environment influenced the proliferation, mobilization, and osteogenic differentiation of ASCs in vitro.The aim of this study was to evaluate the effects of experiment periodontitis on the proliferation, wound healing and osteogenesis markers of adipose-derived stem cells (ASCs) in rats. Materials and methods: Ten male rats were divided into two groups randomly. The control (Con) group received a standard diet, and the periodontitis (Peri) group was received a standard diet with placing ligatures around the maxillary first molar. Toll like receptor 4 (TLR4), Tumor necrosis factor-α (TNF-a) and Interleukin-1β (IL-1β) were tested by immunohistochemistry (IHC) staining and quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation rate of ASCs was measured through Cell Counting Kit-8 (CCK-8) assay. The migration speed of stem cells was evaluated by using a wound healing assay. The expression of alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2) and runt related transcription factor 2 (Runx2) was evaluated by qRT-PCR analysis and western blot. Graph Pad Primer 7.0 software was used for statistical analysis.Results: After 4 weeks, periodontitis model was successfully constructed. The results of IHC and RT-PCR found that in the Peri group, the TNF-a and IL-1β levels of adipose tissues decreased compared with the Con group (P<0.05). The proliferation of Peri-ASCs significantly increased compared with Con-ASCs. Moreover, the wound healing ability of Peri-ASCs gradually increased in a time dependent manner compared with Con-ASCs. Results of RT-PCR showed that ALP and BMP2 gene levels of Peri-ASCs significantly decreased (P<0.05), while the Runx2 gene level in Peri-ASCs was increased, when compared to Con-ASCs. The ALP activity of Peri-ASCs was decreased compared to the Con-ASCs, especially the difference was significant at day 5 day (P<0.01). Western blot results showed that ALP, Runx2 and BMP2 protein levels of Peri-ASCs were significantly lower than those in Con-ASCs after osteogenic induction. Conclusion: Our study demonstrated that experiment periodontitis decreased the expression of TNF-a and IL-1β in adipose tissue in rats. Experiment periodontitis promoted the proliferation and wound-healing ability of ASCs, but obviously inhibited the osteogenic differentiation of ASCs.

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The effect of aging on the biological and immunological characteristics of periodontal ligament stem cells

10.21203/rs.3.rs-17276/v3 ◽

2020 ◽

Author(s):

Xiaoyu Li ◽

Bowen Zhang ◽

Hong Wang ◽

Xiaolu Zhao ◽

Zijie Zhang ◽

...

Keyword(s):

Stem Cells ◽

Periodontal Ligament ◽

Chondrogenic Differentiation ◽

Annexin V ◽

Cell Counting ◽

Blue Staining ◽

Alcian Blue Staining ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Differentiation Potential

Abstract Background: Periodontal ligament stem cells (PDLSCs) have many applications in the field of cytotherapy, tissue engineering, and regenerative medicine. However, the effect of age on the biological and immunological characteristics of PDLSCs remains unclear. Methods: In this study, we compared PDLSCs isolated from young and adult individuals. PDLSCs proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining, and apoptosis level was detected by Annexin V-PE/7-ADD staining. PDLSCs osteogenic/adipogenic/chondrogenic differentiation potentials were assessed by alkaline phosphatase (ALP), Alizarin Red, Oil Red O, Alcian Blue staining and related quantitative analysis. PDLSCs immunosuppressive capacity was determined by EdU and Annexin V-PE/7-ADD staining. To explore its underlying mechanism, microarray, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot analyses were performed to detect differentially expressed genes and proteins in PDLSCs. Results: Our results demonstrated that with aging, the proliferation and osteogenic/adipogenic/chondrogenic differentiation potential of PDLSCs decreased, whereas apoptosis of PDLSCs increased. Moreover, the immunosuppressive ability of PDLSCs decreased with aging. Compared with PDLSCs from young subjects, analysis of mRNA expression revealed an upregulation of CCND3 and RC3H2, and a downregulation of Runx2, ALP, COL1A1, PPARγ2, CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 in those from adult individuals. Furthermore, protein expression levels of Runx2, ALP, COL1A1, and PPARγ2 in the adult group was decreased, whereas that of CCND3 increased. Conclusions: Taken together, aging influences biological and immunological characteristics of PDLSCs, and thus it is more appropriate to utilized PDLSCs from young individuals for tissue regeneration, post-aging treatment, and allotransplantation.

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TGF-β1 is Involved in Vitamin D-Induced Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Regulating the ERK/JNK Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000479997 ◽

2017 ◽

Vol 42 (6) ◽

pp. 2230-2241 ◽

Cited By ~ 14

Author(s):

Xiaorui Jiang ◽

Botao Huang ◽

Huiying Yang ◽

Guishi Li ◽

Chunlei Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Vitamin D ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Chondrogenic Differentiation ◽

Jnk Pathway ◽

Qrt Pcr ◽

Tgf Β1

Background/Aims: Osteoarthritis (OA) is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1) in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

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Chondrogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Chitosan Film Culture

Cell Transplantation ◽

10.3727/096368916x693464 ◽

2017 ◽

Vol 26 (3) ◽

pp. 417-427 ◽

Cited By ~ 17

Author(s):

Tsai-Jung Lu ◽

Fang-Yao Chiu ◽

Hsiao-Ying Chiu ◽

Ming-Chau Chang ◽

Shih-Chieh Hung

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Chondrogenic Differentiation ◽

Chitosan Film ◽

Cartilage Tissue ◽

Blue Staining ◽

Cartilage Defects ◽

Induction Medium ◽

Alcian Blue Staining ◽

Film Culture

Articular cartilage has a very limited capacity for self-repair, and mesenchymal stem cells (MSCs) have the potential to treat cartilage defects and osteoarthritis. However, in-depth mechanistic studies regarding their applications are required. Here we demonstrated the use of chitosan film culture for promoting chondrogenic differentiation of MSCs. We found that MSCs formed spheres 2 days after seeding on dishes coated with chitosan. When MSCs were induced in a chondrogenic induction medium on chitosan films, the size of the spheres continuously increased for up to 21 days. Alcian blue staining and immunohistochemistry demonstrated the expression of chondrogenic proteins, including aggrecan, type II collagen, and type X collagen at 14 and 21 days of differentiation. Importantly, chitosan, with a medium molecular weight (size: 190–310 kDa), was more suitable than other sizes for inducing chondrogenic differentiation of MSCs in terms of sphere size and expression of chondrogenic proteins and endochondral markers. We identified that the mechanistic target of rapamycin (mTOR) signaling and its downstream S6 kinase (S6K)/S6 were activated in chitosan film culture compared to that of monolayer culture. The activation of mTOR/S6K was continuously upregulated from days 2 to 7 of differentiation. Furthermore, we found that mTOR/S6K signaling was required for chondrogenic differentiation of MSCs in chitosan film culture through rapamycin treatment and mTOR knockdown. In conclusion, we showed the suitability of chitosan film culture for promoting chondrogenic differentiation of MSCs and its potential in the development of new strategies in cartilage tissue engineering.

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LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00269-6 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Xiaozhong Zhou ◽

Wangyang Xu ◽

Yeyang Wang ◽

Hui Zhang ◽

Li Zhang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Chondrogenic Differentiation ◽

Genetic Regulation ◽

Luciferase Reporter ◽

Blue Staining ◽

Alcian Blue Staining ◽

Differentiation Markers ◽

Hypoxic Conditions

Abstract Background Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown. Methods rMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2. Results MiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2. Conclusions Our study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

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ID: 1076 Establishing an artificial cartilage from rabbit bone marrow derived mesenchymal stem cell and polycaprolactone scaffold

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.350 ◽

2017 ◽

Vol 4 (S) ◽

pp. 161

Author(s):

Thuy Thi Thanh Dao ◽

Liem Hieu Phan ◽

Ha Thi Ngan Le ◽

Lan Thi Phi ◽

Phuc Van Pham ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Alcian Blue ◽

Chondrogenic Differentiation ◽

Collagen Type ◽

Blue Staining ◽

Type I ◽

Artificial Cartilage ◽

Differentiation Medium ◽

Rabbit Bone

Nowaday, the regenerative medicine based on the use of biomaterial scaffold combine to stem cells was considered as a necessary treatment strategy for injury or diseases involving cartilage. In this study, we investigated the adherence, proliferation and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (rBM-MSCs) on biodegradable polycaprolactone scaffold (PCL). MSCs were isolated from bone marrow of rabbit. rBM-MSC (2x105cell/scaffold) were seeded on PCL and collagen-coated PCL (PCL/col) scaffolds. The cell adhesion and proliferation abilities on the PCL and PCL/col scaffolds were compared to each other to find the better using MTT assay. Then 3D cultured-cells were induced in chondrogenic differentiation medium. The change of scaffold surface structure was evaluated by scanning electron microscope (SEM). The accumulation of aggrecan protein was detected by Alcian Blue staining. The chondrogenic-related gene expression such as sox9, collagen type 1, collagen type 2, collagen type 10, aggrecan, runx2 was assessed by RT-PCR. The existence of artificial cartilage after transplantation of chondrocyte-seeded scaffold under rabbit skin was evaluated by staining histological sections with Alcian Blue. The results showed that rBM-MSCs attached and grown on PCL/col and PCL scaffolds. However, the adhesion efficient of rBM-MSC on PCL/col scaffold was better than PCL scaffolds. Therefore, PCL/col scaffold was used for next experiments. The morphology of rBM-MSCs on PLC/col scaffold were changed and the aggrecan accumulation increased after cultured in chondrogenic differentiation medium. The scaffold surface modification was detected with the presentation of collagen-like fiber structures. The expression of chondrogenesis-associated genes increased after 3, 7, 21, 28 days of chondrogenic induction, especially for collagen type II, collagen type I, aggrecan and collagen type X. Transplanted artificial cartilage was existed in rabbit for six weeks. The mature chondrocyte-like cells were detected and cartilage specific-proteoglycan accumulated more in differentiated cell-scaffold than scaffold seeded undifferentiated-cells. Thus artificial cartilage could be created when differentiating rBM-MSC into chondrocyte on collagen-coated PCL scaffold.

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Survey differentiation of mesenchymal stem cell to chondrocyte using synovial fluid and coldatmospheric plasma treated nanofibrous pcl substrate

MOJ Biology and Medicine ◽

10.15406/mojbm.2021.06.00129 ◽

2021 ◽

Vol 6 (2) ◽

pp. 52-56

Author(s):

Saleh Soleimani ◽

Habibollah Nazem ◽

Mohammad Fazilati ◽

Mohammad Pezeshki Modarres ◽

Seyed Mohammad Atyabi

Keyword(s):

Synovial Fluid ◽

Alcian Blue ◽

Mtt Assay ◽

Chondrogenic Differentiation ◽

Nanofibrous Scaffold ◽

Atmospheric Plasma ◽

Blue Staining ◽

Alcian Blue Staining ◽

Human Mscs ◽

Cold Atmospheric Plasma

Tissue engineering has developed strategy for the repair and regeneration of a variety of tissues. The demand for effective treatment strategies of cartilage cause to autologous human Mesenchymal stem cells (hMSC) used for the repair of cartilage defects. In this study effect of some factors on human MSCs for differentiation to chondrocyte were surveyed such as Present of Poly Ɛ-caprolactone (PCL) nanofibrous scaffold and cold atmospheric plasma (CAP), also effect and amount of synovial fluid were evaluated. In this experimental study cell culture for differentiation assay performed in 6 well pellets by DMEM high glucose medium with a supplement of 10% FBS and synovial fluid 5% and 10% of medium add to pellets for 21 days. Total cellular RNA extracted was used for analysis by RT-PCR. Alcian blue staining confirmed the chondrogenic differentiation. MTT assay results in this study showed that the cells proliferation gradually increased from 1st day to 21st days on the electrospun nanofibrous scaffold. Cellular attachment and proliferation of hMSCs showed higher rate on PCL substrate, modiﬁed with Helium cold atmospheric plasma, compared to the unmodiﬁed and control group at the ﬁrst day to 21st days. Chondrogenic differentiation confirmed by alcian blue staining, and show high degree of conversion to the chondrocyte. MTT assay and the expression of chondrogenic specific genes Collagen type П and Has-2 in comparison with control groups demonstrated cell survival ability, proliferation and cell differentiation by synovial fluid.

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