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In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel 总被引:2,自引:0,他引:2
Yokoyama A Sekiya I Miyazaki K Ichinose S Hata Y Muneta T 《Cell and tissue research》2005,322(2):289-298
Graft implantation is one of the more popular procedures for repairing cartilage defects; however, sacrifices of the donor
site have been an issue. Mesenchymal stem cells (MSCs) are a fascinating source for regenerative medicine because they can
be harvested in a less invasive manner and are easily isolated and expanded, with multipotentiality including chondrogenesis.
MSCs can be isolated from various adult mesenchymal tissues including synovium. Here, we attempted to form cartilage from
the composites of synovium-derived MSCs with collagen gel in vitro. After 21 days of culture, the composites had increased
their cartilage matrix, as demonstrated by toluidine blue staining and immunohistochemistry for type II collagen. The composites
consisting of 5×107 and 108 cells/ml in gel were richer in proteoglycans than those consisting of lower cell densities. After 1 day, MSCs/gel composites
contracted and the diameter decreased by 30%; however, they were stable thereafter. Round cells with short processes producing
collagen fibrils showing a similar morphology to that of chondrocytes were seen in the composites by transmission electron
microscopy. During composite culture, chondroitin sulfate and mRNA expression for cartilage-related genes increased, demonstrating
cartilage maturation. Using an optimized method, we obtained cartilage discs with a diameter of 7 mm and a thickness of 500 μm.
Our procedure should thus make it possible to produce a large cartilage matrix in vitro. The tissue engineering of autologous
cartilage from the composites of synovium-derived MSCs with collagen gel in vitro for transplantation may be a future alternative
to graft implantation for patients with cartilage defects.
This study is supported in part by grants from the Japanese Society for the Promotion of Science (16591478), the Japanese
Orthopaedics and Traumatology Foundation, and the Nakatomi Foundation to I.S., and the Japanese Society for the Promotion
of Science (16591477), the Japanese Sports Medicine Foundation, the Japanese Latest Osteoarthritis Society, and the Center
of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical
and Dental University to T.M. 相似文献
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Tatiana Carla Tomiosso Wilson Romero Nakagaki Laurecir Gomes Stephen Hyslop Edson Rosa Pimentel 《Cell and tissue research》2009,337(2):235-242
The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely,
with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric
oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles
tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was
partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior
to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were
excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also
processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized
by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more
homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast,
in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats
had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon
healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen.
T.C.T. and W.R.N were supported by studentships from CAPES, and S.H. was supported by a research fellowship from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq). 相似文献
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Comparison of rat mesenchymal stem cells derived from bone marrow,synovium, periosteum,adipose tissue,and muscle 总被引:18,自引:0,他引:18
Yoshimura H Muneta T Nimura A Yokoyama A Koga H Sekiya I 《Cell and tissue research》2007,327(3):449-462
Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human
bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of
experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose,
and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each
population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold
higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest
in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets
derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues,
indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages.
The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells.
Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation
potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells
had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a
rat model.
This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence
Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University
(to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic
protein-2 was kindly provided by Astellas Pharma. 相似文献
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Allogeneic mesenchymal stem cells (MSCs) are regarded as promising seed cells for engineering cartilage. However, few researches have covered the immune properties of seeded MSCs. Collagen has been considered as good scaffold, whether it has inherent chondrogenic inducibility for MSCs is still in debate. In this study, engineering grafts are constructed by neonatal rabbit MSCs and collagen Type I hydrogel. After periods of culture, the appearance of chondroid tissue in the grafts and the cartilage matrix‐specific genes expressions of seeded cells prove the inducibility of collagen hydrogel, even if the growth factors are absence. With the differentiation, immunological properties of MSCs are changing. The expressions of main histocompatibility complex (MHC) molecules increase and the ability to inhibit the proliferation of activated lymphocytes may be declined. But to a large extent, it keeps the low stimulating to allogeneic lymphocytes and the small absolute value of MHCs. The changes are adverse for avoiding inflammation and rejection. Therefore, suitable scaffold and engineering strategies should be selected. For the grafts based on Collagen I hydrogel and MSCs, a longer culture period might not be necessary. To maintain the immune regulation, a higher initial MSCs density in engineering grafts may be more meaningful. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
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Uterine receptivity is prerequisite for the attachment of the embryo to the uterine epithelium and involves a specialized polarity-dependent property of uterine epithelial (UE) cells. These UE cells, when polarized in culture, behave like cells in utero by exhibiting apico-basal polarity. In order to develop an implantation model in vitro, UE cells were polarized on extracellular matrix (ECM), and polarity was validated by response to estradiol-17β administered exogenously. UE cells of pregnant rats at day-3 and day-4 post-coitum (p.c.) and of non-pregnant rats were cultured on bare and extracellular-matrix-coated petri dishes until confluency. Hatched blastocysts were transferred to the cultures, and adhesion was monitored every 24 h. Although blastocysts attached to UE cells that were taken from non-pregnant rats and from rats of day-3 p.c. and cultured on bare plastic, they failed to attach to these cells polarized on ECM. However, blastocysts attached firmly to UE cells that had been taken from rats of day-4 p.c. and polarized on ECM. Receptivity of UE cells taken from non-pregnant and pregnant (day-4 p.c.) rats was quantitated by flow cytometric estimation of cellular levels of β3 integrin. The expression of β3 integrin in UE cells from rats of day-4 p.c. was highly significant (P<0.01) when compared with its expression in UE cells from non-pregnant rats. The expression of β3 integrin in UE cells of day-4 p.c. confirmed the receptivity of these cells to blastocyst implantation. Uterine receptivity was also validated in vivo by inducing the decidual cell reaction in rats ovariectomized on day-3 and day-4 p.c. Whereas remarkable deciduoma was noticed in the animals of day-4 p.c., it was absent in the animals of day-3 p.c., thereby indicating that the uterus was receptive on day-4 p.c. only. Thus, blastocysts do not attach to polarized UE cells that have been obtained from a non-receptive uterus. Attachment will occur only if the cells are obtained from a receptive uterus. UE cell receptivity is therefore essential for mimicking the process of implantation in vitro.The authors are grateful to the Ministry of Health and Family welfare, Government of India, for financial support 相似文献
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Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament. 相似文献
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Wenjia Liu Anna Konermann Tao Guo Andreas Jäger Liqiang Zhang Yan Jin 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.Methods
PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.Results
PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.Conclusions
Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.General significance
These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway. 相似文献9.
The current study explored the feasibility and efficacy of co-transfection of the human nerve growth factor (NGF) and vascular endothelial growth factor 165 (VEGF165) genes in rat bone marrow mesenchymal stem cells (BMSCs). The obtained hNGF and vascular endothelial growth factor (VEGF) cDNAs were cloned into the pEGFP-C1 expression vector to construct the recombinant vectors. Co-transfection in rat BMSCs was performed and the expressions of both genes were detected by RT-PCR, Western blot, and enzyme-linked immunospecific assay. The biological activity of recombinant NGF and VEGF proteins was confirmed using the Chick Chorioallantoic Membrane (CAM) assay. NGF and VEGF genes could be expressed successfully in rat BMSCs. The recombinant NGF and VEGF from the rat BMSCs showed a more significant synergetic biological activity compared with single recombinant NGF or VEGF. These findings demonstrate that the co-transfection of hNGF + VEGF genes can enhance the angiogenic effect in vivo. 相似文献
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Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat
Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences 相似文献
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Summary The development of calcitonin cells (C-cells) was investigated in rat thyroid glands from birth to 120 days, using an immunoperoxidase technique and a point-counting method. The proportion of C-cells to follicular cells was 4.5% on the day of birth and increased progressively to 10.4% by 120 days. The highest density of C-cells was noted in the mid-region of the lobes along a longitudinal axis. The caudal and cephalic regions of the lobes contained smaller numbers of C-cells. The C-cells tended to be more numerous in the posterior aspects of the lobes. Although the numbers of C-cells in 120-day-old animals were markedly increased as compared to animals at the time of birth, the cell distributions within the glands were similar at all ages. 相似文献
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Neshati Z Bahrami AR Eshtiagh-Hosseini H Matin MM Housaindokht MR Tabari T Edalatmanesh MA 《Cytotechnology》2012,64(5):485-495
Recent studies have shown that the use of biomaterials and new biodegradable scaffolds for repair or regeneration of damaged tissues is of vital importance. Scaffolds used in tissue engineering should be biodegradable materials with three-dimensional structures which guide the growth and differentiation of the cells. They also tune physical, chemical and biological properties for efficient supplying of the cells to the selected tissues and have proper porosity along with minimal toxic effects. In this manner, the study of these characteristics is a giant stride towards scaffold design. In this study, Gelatin/Siloxane/Hydroxyapatite (GS-Hyd) scaffold was synthesized and its morphology, in vivo biodegradability, cytotoxic effects and ability for cell adhesion were investigated using mesenchymal stem cells (MSCs). The cells were treated with different volumes of the scaffold suspension for evaluation of its cytotoxic effects. The MSCs were also seeded on scaffolds and cultured for 2 weeks to evaluate the ability of the scaffold in promoting of cell adhesion and growth. To check the biodegradability of the scaffold in vivo, scaffolds were placed in the rat body for 21 days in three different positions of thigh muscle, testicle, and liver and they were analyzed by scanning electron microscopy (SEM) and weight changes. According to the results of the viability of this study, no cytotoxic effects of GS-Hyd scaffold was found on the cells and MSCs could adhere on the scaffold with expanding their elongations and forming colonies. The rate of degradation as assessed by weight loss was significant within each group along with significant differences between different tissues at the same time point. SEM micrographs also indicated the obvious morphological changes on the surface of the particles and diameter of the pores through different stages of implantation. The greatest amount of degradation happened to the scaffold particles implanted into the muscle, followed by testicle and liver, respectively. 相似文献
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目的探讨骨髓间充质干细胞(BMSCs)移植对急性肝功能衰竭(ALF)大鼠肝组织中miRNA-155和TNF-α表达的影响,以及与BMSCs疗效间的关系。方法将SD大鼠随机分为健康对照组、ALF组、BMSCs治疗组和BMSCs预防组,其中ALF组予以900 mg/kg D-GalN+10μg/kg脂多糖腹腔注射建立模型;BMSCs治疗组在900 mg/kg D-GalN+10μg/kg脂多糖腹腔注射后2 h,予以尾静脉注射BMSCs 5.0×10^6;BMSCs预防组在900 mg/kg D-GalN+10μg/kg脂多糖腹腔注射前予以尾静脉注射BMSCs 5.0×10^6;健康对照组予以0.9﹪氯化钠溶液1 ml腹腔注射。给药7 h后每组处死大鼠,检测大鼠血清ALT和AST,ELISA法检测TNF-α水平,实时定量PCR检测肝组织miRNA-155、TNF-αmRNA。各组间肝功指标差异采用方差分析,同时观察每组大鼠的24 h生存率,并用卡方检验比较各组生存率的差异。结果 D-GalN/脂多糖诱导7 h后,与ALF组相比,BMSCs预防和BMSCs治疗组大鼠ALT、AST、TNF-α水平均有所降低(P〈0.01);同时两组肝组织TNF-αmRNA和miRNA-155表达水平均有下调(P〈0.01);但两组间相比较差异无统计学意义。ALF组大鼠肝组织miRNA-155上调和TNF-αmRNA诱导呈正相关(r=0.734,P=0.001)。BMSCs预防组和BMSCs治疗组miRNA-155和TNF-αmRNA的部分逆转亦呈正相关(r值分别为0.687和0.590,P值分别为0.004和0.006)。给药后24 h,健康对照组、ALF组、BMSCs治疗组和BMSCs预防组大鼠死亡率组间比较差异有统计学意义(c2=19.078,P〈0.01)。结论在BMSCs干预大鼠ALF发病过程中,可以部分逆转上调的肝组织miRNA-155和TNF-α,且存在协同性,提示BMSCs治疗ALF可能通过对肝组织miRNA-155和TNF-α的调控发生作用。 相似文献
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Mei YF Yamaza T Atsuta I Danjo A Yamashita Y Kido MA Goto M Akamine A Tanaka T 《Cell and tissue research》2007,328(1):117-127
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We investigated the expression of formyl peptide receptor (FPR) and its functional role in human bone marrow-derived mesenchymal stem cells (MSCs). We analyzed the expression of FPR by using ligand-binding assay with radio-labeled N-formyl-met-leu-phe (fMLF), and found that MSCs express FPR. FMLF stimulated intracellular calcium increase, mitogen-activated protein kinases activation, and Akt activation, which were mediated by G(i) proteins. MSCs were chemotactically migrated to fMLF. FMLF-induced MSC chemotaxis was also completely inhibited by pertussis toxin, LY294002, and PD98059, indicating the role of G(i) proteins, phosphoinositide 3-kinase, and extracellular signal regulated protein kinase. N-terminal fragment of annexin-1, Anx-1(2-26), an endogenous agonist for FPR, also induced chemotactic migration of MSCs. Thus MSCs express functional FPR, suggesting a new (patho)physiological role of FPR and its ligands in regulating MSC trafficking during induction of injured tissue repair. 相似文献
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Summary Functional interactions between mast cells and peripheral nerves may occur at sites of association seen in vivo. To study the interactions, we developed a tissue culture model of murine sympathetic neurons co-cultured with rat basophilic leukaemia (RBL-2H3) cells (homologues of mucosal mast cells) or rat peritoneal mast cells. In co-cultures of up to 3 days, light microscopy identified neurite contacts with peritoneal mast cells or RBL-2H3 cells, but not with glial cells or fibroblasts. Electron microscopy confirmed membrane-membrane contact between neurites and RBL-2H3 cells. Time-lapse analysis of interactions between neurons and RBL-2H3 cells showed that 60–100% of the cells in a given field acquired neurite contact within 17 h. In matching control studies, there was no increase in the frequency of neurite contact with cells of the rat plasmacytoma line (YB2/0): these were not selected as targets, and contacts were broken if formed. Time-lapse records of the derivation of neurites from their path suggested a neurotropic effect of mast cells, with neurite contact ensuing when the intervening distance was less than 36±4 m. Once formed, contacts were invariably maintained throughout the period of examination (up to 72 h), in contrast to YB2/0 or fibroblast contacts. We conclude that neurons selectively form and maintain connections with cells representative of rat connective tissue-type and mucosal mast cells in vitro. Similar interactions in vivo could promote nerve/mast cell contacts, which may allow bidirectional communication between the nervous and immune systems. 相似文献
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Mesenchymal stem cells (MSCs) of nonembryortic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem ceils, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide historic H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways,cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency. 相似文献