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Pellet Culture (pellet + culture)
Selected AbstractsPellet culture elicits superior chondrogenic redifferentiation than alginate-based systemsBIOTECHNOLOGY PROGRESS, Issue 4 2009Peter Bernstein Abstract Although pellet culture and encapsulation of chondrocytes into gel-like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate-fibrin, alginate-chitosan) and how do the cells behave in comparison to biomaterial-free (pellet) three-dimensional culturing? Monolayer-expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate-chitosan, alginate-fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate-fibrin,encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate- and alginate-chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D-cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate-cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D-environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Hypertrophy and physiological death of equine chondrocytes in vitroEQUINE VETERINARY JOURNAL, Issue 6 2007Y. A. Ahmed Summary Reasons for performing study: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. Objective: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. Methods: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. Results: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. Conclusions: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. Potential relevance: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis. [source] Development of selective tolerance to interleukin-1, by human chondrocytes in vitro,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Greta M. Lee Interleukin-1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1,. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1, (0.05,10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1, declined over the pretreatment period. In response to IL-1, (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1, on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1,-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1, increased glucose consumption and lactate production throughout the treatment. IL-1, stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE2 and MMP-13 in response to IL-1, than OA cultures. Like the OA, IL-1,-induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL-1,, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP-13 production, metabolic activity, or matrix metabolism. © 2002 Wiley-Liss, Inc. [source] Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2010Tadanao Funakoshi Abstract Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:716,725, 2010 [source] Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2009Shinsuke Sakai Abstract The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01,±,3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27,±,3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 517,521, 2009 [source] Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium,derived mesenchymal stem cellsARTHRITIS & RHEUMATISM, Issue 5 2009Yoichi Ezura Objective Human synovium,derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium,derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis. Methods Human synovium,derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription,polymerase chain reaction. Methylation status was also examined in human articular cartilage. Results Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture. Conclusion The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium,derived MSCs. [source] The influence of sex on the chondrogenic potential of muscle-derived stem cells: Implications for cartilage regeneration and repairARTHRITIS & RHEUMATISM, Issue 12 2008Tomoyuki Matsumoto Objective To explore possible differences in muscle-derived stem cell (MDSC) chondrogenic differentiation in vitro and articular cartilage regeneration in vivo between murine male MDSCs (M-MDSCs) and female MDSCs (F-MDSCs). Methods Three different populations of M- and F-MDSCs (n = 3 of each sex) obtained via preplate technique, which separates cells based on their variable adhesion characteristics, were compared for their in vitro chondrogenic potential using pellet culture. Cells were assayed with and without retroviral transduction to express bone morphogenetic protein 4 (BMP-4). The influence of both expression of stem cell marker Sca1 and in vitro expansion on the chondrogenic potential of M- and F-MDSCs was also determined. Additionally, BMP-4,transduced M- and F-MDSCs were applied to a full-thickness articular cartilage defect (n = 5 each) on the femur of a nude rat, and the quality of the repaired tissue was evaluated by macroscopic and histologic examination. Results With and without BMP-4 gene transduction, M-MDSCs produced significantly larger pellets with a richer extracellular matrix, compared with F-MDSCs. Sca1 purification influenced the chondrogenic potential of MDSCs, especially M-MDSCs. Long-term culture did not affect the chondrogenic potential of M-MDSCs but did influence F-MDSCs. M-MDSCs repaired articular cartilage defects more effectively than did F-MDSCs at all time points tested, as assessed both macroscopically and histologically. Conclusion Our findings demonstrate that sex influences the chondrogenic differentiation and articular cartilage regeneration potential of MDSCs. Compared with female MDSCs, male MDSCs display more chondrogenic differentiation and better cartilage regeneration potential. [source] Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cellsARTHRITIS & RHEUMATISM, Issue 5 2008Michael B. Mueller Objective Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers. Methods Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor , (TGF,), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription,polymerase chain reaction. Results The combination of TGF, withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1,3, parathyroid hormone,related protein receptor, retinoic acid receptor ,, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed. Conclusion Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering. [source] Inhibition of cartilage degradation: A combined tissue engineering and gene therapy approachARTHRITIS & RHEUMATISM, Issue 3 2003Wael Kafienah Objective To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. Methods Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1,transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. Results Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1,transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1,transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. Conclusion Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans. [source] Pellet culture elicits superior chondrogenic redifferentiation than alginate-based systemsBIOTECHNOLOGY PROGRESS, Issue 4 2009Peter Bernstein Abstract Although pellet culture and encapsulation of chondrocytes into gel-like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate-fibrin, alginate-chitosan) and how do the cells behave in comparison to biomaterial-free (pellet) three-dimensional culturing? Monolayer-expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate-chitosan, alginate-fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate-fibrin,encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate- and alginate-chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D-cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate-cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D-environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Carboxypeptidase Z (CPZ) Links Thyroid Hormone and Wnt Signaling Pathways in Growth Plate Chondrocytes,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2009Lai Wang Abstract Carboxypeptidase Z (CPZ) removes carboxyl-terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine-rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt-4 expression and Wnt/,-catenin signaling. The Wnt-4 protein contains a C-terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/,-catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt-4 mRNA are co-expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt-4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt-4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/,-catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C-terminal arginine residue from a synthetic peptide consisting of the carboxyl-terminal 16 amino acids of the Wnt-4 protein. Removal of the C-terminal arginine residue of Wnt-4 by site-directed mutagenesis enhances the positive effect of Wnt-4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ-enhanced activation of Wnt-4. [source] Development of selective tolerance to interleukin-1, by human chondrocytes in vitro,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Greta M. Lee Interleukin-1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1,. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1, (0.05,10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1, declined over the pretreatment period. In response to IL-1, (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1, on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1,-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1, increased glucose consumption and lactate production throughout the treatment. IL-1, stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE2 and MMP-13 in response to IL-1, than OA cultures. Like the OA, IL-1,-induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL-1,, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP-13 production, metabolic activity, or matrix metabolism. © 2002 Wiley-Liss, Inc. [source] Multilineage mesenchymal differentiation potential of human trabecular bone-derived cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2002Ulrich Nöth Abstract Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-,1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated prote-oglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, ,-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor ,2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-l-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responsesARTHRITIS & RHEUMATISM, Issue 9 2009Shigeru Miyaki Objective MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). Methods To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1, (IL-1,) on miR-140 expression. Double-stranded miR-140 (ds,miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. Results Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1, suppressed miR-140 expression. Transfection of chondrocytes with ds,miR-140 down-regulated IL-1,,induced ADAMTS5 expression and rescued the IL-1,,dependent repression of AGGRECAN gene expression. Conclusion This study shows that miR-140 has a chondrocyte differentiation,related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1, may contribute to the abnormal gene expression pattern characteristic of OA. [source] Blocking vascular endothelial growth factor with soluble Flt-1 improves the chondrogenic potential of mouse skeletal muscle,derived stem cellsARTHRITIS & RHEUMATISM, Issue 1 2009Seiji Kubo Objective To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle. Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real-time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP-4) were coimplanted with MDSCs transduced to express either VEGF or sFlt-1 (a VEGF antagonist) to provide a gain- and loss-of-function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt-1,treated) pellet cultures, but quantitative PCR revealed that sFlt-1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP-4 and transforming growth factor ,3 (TGF,3). In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt-1 improved the MDSC-mediated repair of articular cartilage, compared with BMP-4 alone. Conclusion Soluble Flt-1 gene therapy improved the BMP-4, and TGF,3-induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage. [source] Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cellsARTHRITIS & RHEUMATISM, Issue 5 2008Michael B. Mueller Objective Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers. Methods Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor , (TGF,), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription,polymerase chain reaction. Results The combination of TGF, withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1,3, parathyroid hormone,related protein receptor, retinoic acid receptor ,, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed. Conclusion Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering. [source] Inhibition of cartilage degradation: A combined tissue engineering and gene therapy approachARTHRITIS & RHEUMATISM, Issue 3 2003Wael Kafienah Objective To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. Methods Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1,transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. Results Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1,transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1,transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. Conclusion Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans. [source] Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritisARTHRITIS & RHEUMATISM, Issue 3 2002J. Mary Murphy Objective Mesenchymal stem cells (MSCs) are resident in the bone marrow throughout normal adult life and have the capacity to differentiate along a number of connective tissue pathways, among them bone, cartilage, and fat. To determine whether functionally normal MSC populations may be isolated from patients with advanced osteoarthritis (OA), we have compared cells from patients undergoing joint replacement with cells from normal donors. Cell populations were compared in terms of yield, proliferation, and capacity to differentiate. Methods MSCs were prepared from bone marrow aspirates obtained from the iliac crest or from the tibia/femur during joint surgery. In vitro chondrogenic activity was measured as glycosaminoglycan and type II collagen deposition in pellet cultures. Adipogenic activity was measured as the accumulation of Nile Red O-positive lipid vacuoles, and osteogenic activity was measured as calcium deposition and by von Kossa staining. Results Patient-derived MSCs formed colonies in primary culture that were characteristically spindle-shaped with normal morphology. The primary cell yield in 36 of 38 cell cultures from OA donors fell within the range found in cultures from normal donors. However, the proliferative capacity of patient-derived MSCs was significantly reduced. There was a significant reduction in in vitro chondrogenic and adipogenic activity in cultures of patient-derived cells compared with that in normal cultures. There was no significant difference in in vitro osteogenic activity. There was no decline in chondrogenic potential with age in cells obtained from individuals with no evidence of OA. Conclusion These results raise the possibility that the increase in bone density and loss of cartilage that are characteristic of OA may result from changes in the differentiation profile of the progenitor cells that contribute to the homeostatic maintenance of these tissues. [source] Cryopreservation and in Vitro Expansion of Chondroprogenitor Cells Isolated from the Superficial Zone of Articular CartilageBIOTECHNOLOGY PROGRESS, Issue 1 2005Juan M. Melero Martin Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 ,L FCS cm,2 h,1 was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 × 105 cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 × 105 cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-,1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time. [source] | |||||||||||||