共查询到20条相似文献,搜索用时 28 毫秒
1.
O. Raabe C. Reich S. Wenisch A. Hild M. Burg-Roderfeld H.-C. Siebert S. Arnhold 《Histochemistry and cell biology》2010,134(6):545-554
Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate
into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming
growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential
of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet
culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for
a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and
type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous
extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG).
Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures
stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the
same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as
it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to
induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue
engineering, especially for cartilage repair. 相似文献
2.
Perrier E Ronzière MC Bareille R Pinzano A Mallein-Gerin F Freyria AM 《Biotechnology letters》2011,33(10):2091-2101
Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage
defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated
their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of
BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing
effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in
expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was
observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate
chondrogenesis with a particular effect on the main cartilage collagens. 相似文献
3.
Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts,
chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased
the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion
of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the
medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’
culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression
levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic
differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction
of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with
FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation
potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found
that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated
by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic
differentiation potential of hMSCs in the presence of FGF-2. 相似文献
4.
The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.This work was supported by grants R92-001-1 and R92-001-2 from the Veterans General Hospital, Taipei, and grant NSC-92-2314-B-075-022 from the National Science Council, Taiwan. 相似文献
5.
6.
Bone-marrow-derived mesenchymal stem cells (MSCs) are candidates for regeneration applications in musculoskeletal tissue such
as cartilage and bone. Various soluble factors in the form of growth factors and cytokines have been widely studied for directing
the chondrogenic and osteogenic differentiation of MSCs, but little is known about the way that the composition of extracellular
matrix (ECM) components in three-dimensional microenvironments plays a role in regulating the differentiation of MSCs. To
define whether ECM components influence the regulation of osteogenic and chondrogenic differentiation by MSCs, we encapsulated
MSCs in poly-(ethylene glycol)-based (PEG-based) hydrogels containing exogenous type I collagen, type II collagen, or hyaluronic
acids (HA) and cultured them for up to 6 weeks in chondrogenic medium containing transforming growth factor-β1 (10 ng/ml)
or osteogenic medium. Actin cytoskeleton organization and cellular morphology were strongly dependent on which ECM components
were added to the PEG-based hydrogels. Additionally, chondrogenic differentiation of MSCs was marginally enhanced in collagen-matrix-based
hydrogels, whereas osteogenic differentiation, as measured by calcium accumulation, was induced in HA-containing hydrogels.
Thus, the microenvironments created by exogenous ECM components seem to modulate the fate of MSC differentiation. 相似文献
7.
Sheehy EJ Buckley CT Kelly DJ 《Biochemical and biophysical research communications》2012,417(1):305-310
The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO(2)) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO(2)). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO(2) proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO(2) also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO(2) was found to be a more potent promoter of chondrogenesis than expansion at 5% pO(2). Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO(2) compared to 5% pO(2). Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO(2) also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a chondrogenic phenotype for use in cartilage repair therapies or to promote hypertrophy of cartilaginous grafts for endochondral bone repair strategies. 相似文献
8.
目的:研究软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法:BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果:质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高(P<0.05)。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原(Col1a1)mRNA水平均显著低于对照组(P<0.05),RUNX2、骨钙蛋白(Osteocalcin)蛋白表达水平明显低于对照组(P<0.05),而成软骨标记基因SOX9、蛋白聚糖(Aggrecan)mRNA水平均显著高于对照组(P<0.05),SOX9、Ⅱ型胶原(Col2a1)蛋白表达均明显多于对照组(P<0.05)。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原(Col10a1)基因表达显著低于对照组(P<0.05),结论:骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。 相似文献
9.
Wanyao Xia Yu-Qing Jin James D. Kretlow Wei Liu Wenlong Ding Hengyun Sun Guangdong Zhou Wenjie Zhang Yilin Cao 《Biotechnology letters》2009,31(5):639-646
TGF-β1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells
(BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the
hTGF-β1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA
scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-β1 expressed
and secreted more hTGF-β1 protein in vitro than those of the control group. Histological and immunohistological examination
of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage
formation after 3 weeks in vivo. The neocartilage occupied 42 ± 5% of the total tissue volume which was significantly greater
than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen
in the AdhTGF-β1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG
in experimental groups. The results demonstrate that transfer of hTGF-β1 into BMSCs via adenoviral transduction can induce
chondrogenic differentiation in vitro and enhance chondrogenesis in vivo. 相似文献
10.
Tetsu Kobayashi Xiangde Liu Hui Jung Kim Tadashi Kohyama Fu-Qiang Wen Shinji Abe Qiuhong Fang Yun Kui Zhu John R Spurzem Peter Bitterman Stephen I Rennard 《Respiratory research》2005,6(1):141
Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury. 相似文献
11.
Kolambkar YM Peister A Soker S Atala A Guldberg RE 《Journal of molecular histology》2007,38(5):405-413
For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible,
can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been
isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of
this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human
AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There
was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-β
(TGF-β) supplementation, with TGF-β1 producing greater increases than TGF-β3. Modification of expansion media supplements
and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-β1 supplementation
alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three
weeks of TGF-β1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate
along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications. 相似文献
12.
Gordeladze JO Noël D Bony C Apparailly F Louis-Plence P Jorgensen C 《Experimental cell research》2008,314(7):1495-1506
In order to ensure that MSCs designed for in vivo cartilage repair do not untowardly differentiate into osteoblasts and mineralize in situ, we tested whether siRNA-induced suppression of cbfa1/Runx2 affected the osteogenic and chondrogenic differentiation potential of the murine cell line C3H10T1/2. Anti-cbfa1/Runx2 siRNA decreased the levels of cbfa1/Runx2 mRNA and protein by 65-80%, and also markedly reduced the expression of osteoblast-related genes such as Dlx5, osterix, collagen type I, alkaline phosphatase (AP), osteocalcin, SPARC/osteonectin and osteopontin, leading to a temporal expression of AP enzyme activity and mineralization potential delayed by at least some 7-9 days. Furthermore, siRNA-transfected cells, grown under chondrogenic conditions did not display biologically significant changes in the expression of aggrecan, collagen type II or type X, or histology when grown in micropellets or monolayer cultures. Finally, when cells were propagated in osteogenic medium and injected into the tibial muscles of SCID mice, no overtly mineralized bone tissue emerged. These experiments indicate that a major transient reduction of cbfa1/Runx2 expression in MSCs is sufficient to delay osteoblastic differentiation, both in vitro and in vivo, while chondrogenesis seemed to be sustained. 相似文献
13.
Bosnakovski D Mizuno M Kim G Takagi S Okumura M Fujinaga T 《Biotechnology and bioengineering》2006,93(6):1152-1163
Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation. 相似文献
14.
Periosteum-derived progenitor cells (PDPCs) could be differentiated into cartilage using atelocollagen as a carrier and in
the presence of transforming growth factor-β3 (TGF-β3). Chondrogenesis was verified by RT-PCR and Western blotting. Expression
of the type II collagen mRNA was found from the differentiated PDPCs in atelocollagen 3 weeks after chondrogenic induction.
The chondrogenic potential of the PDPCs was also verified by histochemical staining for type II collagen protein. Increased
production of glycosaminoglycan shows that the PDPCs in atelocollagen could differentiate into chondrocytes under a chondrogenic
environment. PDPCs can therefore be used as a cell source for cell-based therapies targeted toward the articular cartilage
of the knee. 相似文献
15.
Conditioned medium from adipose derived stem cells (ADSC-CM) stimulates both collagen synthesis and migration of fibroblasts,
and accelerates wound healing in vivo. Recently, the production and secretion of growth factors has been identified as an
essential function of adipose-derived stem cells (ADSCs). However, the main soluble factor of ADSC-CM which mediates paracrine
effects and its underlying mechanism has not been elucidated yet. In this study, we considered transforming growth factor-beta1
(TGF-β1) as a strong candidate for paracrine effect of ADSC-CM and investigated collagen synthesis and hyaluronic acid synthase
(HAS) expression. After ADSC-CM addition, collagen type I, type III, HAS and hyaluronic acid (HA) expressions on human dermal
fibroblasts (HDFs) were evaluated. Furthermore, to clarify effects of TGF-β1 as a paracrine mediator, TGF-β1 antibody and
external supplementary TGF-β1 were treated to HDFs. Collagens type I, type III, HAS-1 and HAS-2 mRNA expressions of HDFs were
greatly increased by ADSC-CM treatment, however there was no change in TGF-β1 antibody treated HDFs compared with non-treated
control. These results strongly demonstrate that TGF-β1 plays an important role as a paracrine mediator of ECM synthesis.
The fact that TGF-β1 contained in ADSC-CM not only accelerates collagen deposition but also increase hyaluronic acid synthesis
of HDFs through HAS-1 and HAS-2 expression was also elucidated in this study. Therefore, ADSC-CM shows promise for the treatment
of cutaneous wounds and accelerates granulation formation during healing process. 相似文献
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18.
Berna Tezcan Sema Serter Esat Kiter A. Cevik Tufan 《Journal of molecular histology》2010,41(4-5):247-258
Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10?8 and 10?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect. 相似文献
19.
Emilie Roeder Christel Henrionnet Jean Christophe Goebel Nicolas Gambier Olivier Beuf Denis Grenier Bailiang Chen Pierre-André Vuissoz Pierre Gillet Astrid Pinzano 《PloS one》2014,9(5)