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Osteogenesis characteristics of cultured rat mesenchymal stem cells under bone induction condition  [cached]
CHEN Dong-Feng
Zhong Xi Yi Jie He Xue Bao , 2004,
Abstract: Objective: To investigate the osteogenesis characteristics of cultured rat mesenchymal stem cells (MSCs) under bone induction condition. Methods: MSCs were isolated from adult rat by using density gradient separation method. The osteogenic inducers were compounds of Dexone, β-glycerophosphate sodium and vitamin C. Results: The MSC attachment formed soon after the seeding and grew into colonies with the appearance of fibroblastic cells. The osteogenic inducer with low dose of Dexone could promote the osteogenic differentiation of MSC.In the group of osteogenic inducer with low dose of Dexone, the expression of alkaline phosphatase (ALP) was remarkably increased after one week’s induction, and the number of positive cells was (15.1±2.6), significantly higher than that of the control group (12.0±3.5) (P<0.01). The calcified deposits began to appear in the group of osteogenic inducer with low dose of Dexone after one week's induction and was increased remarkably after three weeks, and the number of calcified deposits was (9.0±1.7), significantly higher than that of the control group (2.0±1.8) (P<0.01).Conclusion: MSC can differentiate into osteogenesis by osteogenic induction and may be used to provide seed cells for bone tissue engineering.
Knockdown of the HDAC1 Promotes the Directed Differentiation of Bone Mesenchymal Stem Cells into Cardiomyocytes  [PDF]
Dong-feng Lu, Ying Wang, Zi-zhuo Su, Zhao-hua Zeng, Xiao-wen Xing, Zhi-yu He, Chunxiang Zhang
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0092179
Abstract: Failure of the directed differentiation of the transplanted stem cells into cardiomyocytes is still a major challenge of cardiac regeneration therapy. Our recent study has demonstrated that the expression of histone deacetylase 1 (HDAC1) is decreased in bone mesenchymal stem cells (BMSCs) during their differentiation into cardiomyocytes. However, the potential roles of HDAC1 in cardiac cell differentiation of BMSCs, as well as the mechanisms involved are still unclear. In current study, the expression of HDAC1 in cultured rat BMSCs is knocked down by lentiviral vectors expressing HDAC1-RNAi. The directed differentiation of BMSCs into cardiomyocytes is evaluated by the expression levels of cardiomyocyte-related genes such as GATA-binding protein 4 (GATA-4), Nirenberg, Kim gene 2 homeobox 5 (Nkx2.5), cardiac troponin T (CTnT), myosin heavy chain (MHC), and connexin-43. Compared with that in control BMSCs, the expression of these cardiomyocyte-related genes is significantly increased in these HDAC1 deficient stem cells. The results suggest that HDAC1 is involved in the cardiomyocyte differentiation of BMSCs. Knockdown of the HDAC1 may promote the directed differentiation of BMSCs into cardiomyocytes.
Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells
Zhang WJ, Li ZH, Huang QF, Xu L, Li JH, Jin YQ, Wang GF, Liu XY, Jiang XQ
International Journal of Nanomedicine , 2013, DOI: http://dx.doi.org/10.2147/IJN.S39357
Abstract: ts of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells Original Research (1099) Total Article Views Authors: Zhang WJ, Li ZH, Huang QF, Xu L, Li JH, Jin YQ, Wang GF, Liu XY, Jiang XQ Published Date January 2013 Volume 2013:8 Pages 257 - 265 DOI: http://dx.doi.org/10.2147/IJN.S39357 Received: 19 October 2012 Accepted: 02 December 2012 Published: 11 January 2013 Wenjie Zhang,1,2,* Zihui Li,3,* Qingfeng Huang,1 Ling Xu,1 Jinhua Li,3 Yuqin Jin,1,2 Guifang Wang,1,2 Xuanyong Liu,2 Xinquan Jiang1 1Department of Prosthodontics, 2Oral Bioengineering Laboratory, Shanghai Research Institute of Stomatology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 3State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, China *These authors contributed equally to this work Background and methods: Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. Results: The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. Conclusion: This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.
Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells  [cached]
Zhang WJ,Li ZH,Huang QF,Xu L
International Journal of Nanomedicine , 2013,
Abstract: Wenjie Zhang,1,2,* Zihui Li,3,* Qingfeng Huang,1 Ling Xu,1 Jinhua Li,3 Yuqin Jin,1,2 Guifang Wang,1,2 Xuanyong Liu,2 Xinquan Jiang11Department of Prosthodontics, 2Oral Bioengineering Laboratory, Shanghai Research Institute of Stomatology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 3State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, China *These authors contributed equally to this workBackground and methods: Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells.Results: The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells.Conclusion: This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.Keywords: micro/nanotexture, nanorod, titanium surface, bone marrow mesenchymal stem cells, adhesion, osteogenic differentiation
Effects of Severe Hypoxia on Bone Marrow Mesenchymal Stem Cells Differentiation Potential  [PDF]
Claudia Cicione,Emma Mui?os-López,Tamara Hermida-Gómez,Isaac Fuentes-Boquete,Silvia Díaz-Prado,Francisco J. Blanco
Stem Cells International , 2013, DOI: 10.1155/2013/232896
Abstract: Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed. 1. Introduction Mesenchymal stem cells (MSCs) are multipotent cells that can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into multiple lineages [1–5]. These cells are located in bone marrow (BM), around blood vessels, in fat, skin, muscle, and other tissues, and their presence contributes to the reparative capacity of these tissues. MSCs from different tissue sources can have biologic distinctions. In this way, MSCs derived from bone marrow show a higher potential for osteogenic differentiation [6], while MSCs of synovial origin show a greater tendency toward chondrogenic differentiation [7]. Moreover, under identical culture conditions of differentiation, MSCs isolated from the synovial membrane show more chondrogenic potential than those derived from bone marrow, periosteum, skeletal muscle, or adipose tissue [8]. The recent use of autologous or allogenic stem cells has been suggested as an alternative therapeutic approach for treatment of cartilage defects [9], with these cells representing a promising resource for different tissue engineering and cell-based therapies
Temporal Expression of Pelp1 during Proliferation and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells  [PDF]
Jing Wang, Shujun Song, Liang Shi, Qiang Zhu, Chuanchuan Ma, Xiaoqing Tan, Yin Ding, Zhongying Niu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075477
Abstract: Background Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1). Objective To investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation. Methods rBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP). Results Over 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media. Conclusions Pelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.
Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells
Yan Huang, Zhong-Quan Dai, Shu-Kuan Ling, Hong-Yu Zhang, Yu-Min Wan, Ying-Hui Li
Journal of Biomedical Science , 2009, DOI: 10.1186/1423-0127-16-87
Abstract: Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.The availability of sufficient, suitable cells is a limiting factor in regenerative medicine or cellular therapy which is a potential method for treatment of some diseases, such as myocardial infarction and bone defects. Mesenchymal stem cells (MSCs) are an attractive source of material for cellular replacement strategies for clinical applications and tissue engineering owing to their ability to replicate in the undifferentiated state, to differentiate into different cell lineages and the low immunogenic response in vivo [1-4]. Many studies have reported that MSCs can differentiate into cardiomyocytes after exposure to 5-azacydine (5-aza) [5,6], into osteoblasts in th
An Oligodeoxynucleotide That Induces Differentiation of Bone Marrow Mesenchymal Stem Cells to Osteoblasts in Vitro and Reduces Alveolar Bone Loss in Rats with Periodontitis  [PDF]
Yuqin Shen,Zhiyuan Feng,Chongtao Lin,Xu Hou,Xueju Wang,Jing Wang,Yongli Yu,Liying Wang,Xinhua Sun
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13032877
Abstract: To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.
Comparison of proliferative and multilineage differentiation potentials of cord matrix, cord blood, and bone marrow mesenchymal stem cells  [cached]
Shetty Prathibha,Cooper Khushnuma,Viswanathan Chandra
Asian Journal of Transfusion Science , 2010,
Abstract: Background: Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the two widely studied and characterized adult stem cells. Thus far, MSCs were obtained from the bone marrow, which is a painful procedure. Therefore, MSCs from less common sources like cord blood, adipose tissue, tooth pulp, and so on, have been the subject of research. The purpose of this study is to explore the possibility of finding MSCs from a less controversial, easy, and abundant source, such as the umbilical cord, for potential regenerative medicine applications. Study Design and Methods: Five bone marrow samples (BM), seventy cord blood units (CB), and four umbilical cord matrix (CM) samples have been used for the study. Expanded MSCs were checked for biomarker expression by flow cytometry and were also checked for their differentiation to mesodermal and ectodermal lineages. Results: MSCs could be isolated from 100% BM and CM samples, as compared to only 6% of CB samples. The fold expansion of the mesenchymal stem cells observed in CB (CB-MSCs) was distinctly higher as compared to BM (BM-MSCs) and CM (CM-MSCs). MSCs isolated from all the three sources expressed a characteristic mesenchymal phenotype of CD45-/vWF-/CD14-/CD31-/CD73+/CD105+/SSEA4+/CD29+/CD44+/HLAABC+, whereas, the HLA DR was conspicuously absent in CM-MSCs and CB-MSCs. Although osteogenic, chondrogenic, and neural differentiation was observed in MSCs from all sources, adipogenic differentiation was observed only in BM-MSCs. Conclusion: CM-MSCs are a dependable source of an unlimited number of MSCs for autologous and allogenic use in regenerative medicine.
Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow
Shu-Wen Qian, Xi Li, You-You Zhang, Hai-Yan Huang, Yuan Liu, Xia Sun, Qi-Qun Tang
BMC Developmental Biology , 2010, DOI: 10.1186/1471-213x-10-47
Abstract: Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse.The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.Increased adipose tissue mass associated with obesity is due to the increased number and size of adipocytes [1,2]. Adipocyte differentiation from mesenchymal stem cells plays an important role in the hyperplasia of adult adipose tissue. A population of cells resident in the vascular stroma of adipose tissue can differentiate into adipocytes in vitro and in vivo [3]. Recent studies indicate that pericytes in blood vessel walls have adipogenic potential, express mesenchymal stem cell (MSC) markers and are multipotent [4]. In addition to resident stem cells, non-resident stem cells can serve as a source of adipocyte precursors; bone marrow MSCs can be recruited to adipose tissue and generate new adipocytes in response to treatment with thiazolidinediones (TZDs) or high fat stimulation [5].The characteristics and molecular mechanism underlying adipocyte differentiation have been extensively investigated in the murine pre-adipocyte cell lines 3T3-L1 and 3T3-F442A [6,7]. Growth-arrested pre-adipocytes have been shown to re-enter the cell cycle synchronously and undergo mitotic clonal expansion in res
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