Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online July 26, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2786 -2796
doi:10.1634/stemcells.2007-0374; www.StemCells.com
© 2007 AlphaMed Press

OPEN ACCESS ARTICLE
This Article
Free via Open Access: OA
Right arrow OA Abstract
Right arrow Full Text (PDF)
Right arrowOA All Versions of this Article:
2007-0374v1
25/11/2786    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Murdoch, A. D.
Right arrow Articles by Hardingham, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murdoch, A. D.
Right arrow Articles by Hardingham, T. E.

TISSUE-SPECIFIC STEM CELLS

Chondrogenic Differentiation of Human Bone Marrow Stem Cells in Transwell Cultures: Generation of Scaffold-Free Cartilage

Alan D. Murdoch, Lisa M. Grady, Matthew P. Ablett, Theoni Katopodi, Roger S. Meadows, Tim E. Hardingham

UK Centre for Tissue Engineering and Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Faculty of Life Sciences, Michael Smith Building, Manchester, United Kingdom

Key Words. Mesenchymal stem cells • Chondrogenesis • Extracellular matrix • Gene expression

Correspondence: Tim E. Hardingham, Ph.D., UK Centre for Tissue Engineering and Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, U.K. Telephone: +44 (0)161 275 5511; Fax: +44 (0)161 275 5082; e-mail: timothy.e.hardingham{at}manchester.ac.uk

Received May 15, 2007; accepted for publication July 16, 2007.
First published online in STEM CELLS EXPRESS   July 26, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high-density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2–4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8–1 mm in thickness from 0.5 x 106 cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Adult human mesenchymal stem cells have been derived from a variety of tissues and have shown the potential to participate in growth and repair processes. Stem or progenitor cells from the bone marrow have been expanded in culture and have been shown to develop into multiple cell lineages in vivo [1–3] and into a variety of anchorage-dependent mesenchymal and other cell types (bone, cartilage, fat, muscle, nerve) in vitro [4–8]. The potential to repair damaged or diseased tissues with an autologous cell source has resulted in a great deal of interest in these cells to provide the basis for strategies in regenerative medicine.

Damaged human articular cartilage has only a limited capacity for repair. The resident cartilage cells, the chondrocytes, fail to mount an effective repair process, and the cartilage appears unable to recruit local sources of progenitor cells at the articular surface [9] and in the synovial lining of the joint cavity [10, 11]. If the subchondral plate underlying the cartilage is penetrated, ingress of cells from the bone marrow, including MSCs, frequently results in a fibrocartilaginous repair [12]. Isolated MSCs from rabbit and human bone marrow were first shown to be capable of in vitro chondrogenic differentiation in micromass pellet cultures using a serum-free defined culture medium [13, 14]. Critical factors in chondrogenic differentiation of MSCs in vitro appear to be the high initial cell density and exposure of the cells to signals from glucocorticoids and members of the transforming growth factor (TGF)β family [13–15]. This system has been used to examine the effects of various soluble factors and signaling pathways on the control of the differentiation process [16–21] and has been adapted to medium/high throughput analysis [22]. Other workers have also employed polymer- or biomaterial-based scaffolds [23–30] or hydrogel supports such as alginates, collagen gels, and agarose [30–33] to produce constructs of larger sizes. Similar constructs utilizing ex vivo expanded autologous MSCs have been used in some human clinical applications [34, 35]. In this report, we show the improved chondrogenic differentiation of human bone marrow stem cells (hMSC) in a Transwell culture on a flexible porous membrane support and provide evidence that it results in more rapid and efficient differentiation and in more synthesis and deposition of cartilage-like matrix than other established chondrogenic protocols.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human Bone Marrow Stem Cell Culture
Human bone marrow stem cells (from five donors 20–44 years of age) were isolated from human bone marrow mononuclear cells (Lonza Biosciences, Berkshire, U.K., http://www.lonza.com) by adherence over 24 hours to tissue culture plastic and were expanded in monolayer culture in Mesenchymal Stem Cell Growth Medium (Lonza) supplemented with 5 ng/ml fibroblast growth factor-2 (R&D Systems Europe, Ltd., Abingdon, U.K., http://www.rndsystems.com). Cultures were maintained in a humid atmosphere of 5% CO2/95% air at 37°C. Once cells had reached confluence (passage 1 [P1]), they were passaged using Trypsin/EDTA at a split ratio of 1:3. Experiments were performed using cells at P3, and all experiments were repeated with cells from 2–5 donors.

Chondrogenic Differentiation
Isolated hMSC were resuspended in chondrogenic culture medium consisting of high glucose Dulbecco's modified Eagle's medium containing 100 µg/ml sodium pyruvate (Lonza), 10 ng/ml TGFβ3 (R&D Systems), 100 nM dexamethasone, 1x ITS+1 premix, 40 µg/ml proline, and 25 µg/ml ascorbate-2-phosphate (all from Sigma-Aldrich, Poole, U.K., http://www.sigmaaldrich.com) [36]. For chondrogenesis in pellet cultures, aliquots of hMSC (5 x 105) were spun in 15-ml polypropylene tubes (1 ml of medium, 240g, 5 minutes) [13, 14, 36]. For chondrogenesis in Transwells, aliquots of hMSC (5 x 105 in 100 µl of medium) were pipetted onto dry 6.5-mm diameter, 0.4-µm pore size polycarbonate Transwell filters (Corning B.V. Life Sciences, Schiphol-Rijk, The Netherlands, http://www.corning.com) and spun in a 24-well plate (200g, 5 minutes). Culture continued in the 24-well plate (Corning Life Sciences) with 0.5 ml of chondrogenic medium added to the lower well, which submerged the membrane and cells. Medium in Transwell and pellet cultures was replaced every second day for up to 28 days.

Biochemical Analysis
Chondrogenic pellets and Transwell discs were digested with papain (10 U/ml) at 60°C [37]. The sulfated glycosaminoglycan (GAG) content was measured by 1,9-dimethylmethylene blue binding (Sigma) [38] using shark chondroitin sulfate (Sigma) as standard, and the DNA content was measured with either Hoechst 33258 (Sigma) or PicoGreen (Invitrogen, Paisley, U.K., http://www.invitrogen.com) intercalating dyes. In some experiments, spent medium was collected at each medium change and the sulfated GAG measured to estimate the total glycosaminoglycan released into the medium during culture. Collagen content was estimated from the hydroxyproline content measured by the method of Bergman and Loxley [39]. Briefly, acid hydrolyzed (6 N HCl, 105°C, 18 hours) dried samples were reacted with chloramine-T and dimethylaminobenzaldehyde with the absorbance measured at 570 nm in a 96-well plate. Hydroxyproline content was determined by comparison with a range of hydroxyproline standards, and the collagen content was calculated based on a hydroxyproline content of 10% (wt/wt). For analysis of major fibrillar collagen {alpha} chains, pellets and discs were digested with bovine testicular hyaluronidase (0.1 mg/ml in phosphate-buffered saline, room temperature, 2 hours) and pepsin (0.1 mg/ml in 0.5 M acetic acid, 4°C, 24–48 hours). Portions of the digests were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (NuPAGE; Invitrogen) and stained with Coomassie Brilliant Blue. The intensities of stained collagenous bands in a range of gel loadings were determined for each sample on a GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hemel Hempstead, U.K., http://www.bio-rad.com) and compared with standard collagen preparations from human placenta or rat tail tendon. The triple helical regions of the {alpha}1 chains of collagens type I and type II (and, under reducing conditions, type III) all comigrate on SDS-PAGE gels but run slower than the {alpha}2(I) chain. Densitometric analysis of the ratio of {alpha}1 band/{alpha}2 band of the major pepsin-resistant proteins by SDS-PAGE (under nonreducing conditions) was therefore used to estimate the relative proportions of collagen II and collagen I. The wet masses of disc and pellet cartilage constructs were determined after being drained of culture medium, and the dry mass of some constructs was determined by desiccation to constant mass in a vacuum oven at 60°C.

Immunohistochemistry, Histology, and Electron Microscopy
Transwell discs and pellets were fixed in 4% paraformaldehyde or HistoChoice (Amresco Inc., Solon, OH, http://www.amresco-inc.com) and processed into paraffin wax. Sections cut at 5 µm were stained with hematoxylin and eosin or Safranin O/Fast Green to reveal polyanion distribution. Other sections were probed with antibodies raised against collagen type II (N19 polyclonal), collagen type I (C18 polyclonal) (both from Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), collagen type X (mouse anti-deer antler type X, kind gift from Dr. Gary Gibson [40, 41]), and aggrecan (rabbit antiserum BR1 [42]). Sections were digested with Chondroitinase ABC (Sigma; 0.1 U/ml, 45 minutes, 37°C) and incubated at 4°C overnight with primary antibodies at an appropriate dilution in block solution. Secondary biotinylated antibody signal was amplified by incubating with streptavidin/peroxidase (StreptABComplex/HRP; Dako UK Ltd., Ely, U.K., http://www.dako.co.uk) and developed using 3,3'-diaminobenzidine (Sigma Fast DAB). Images were collected using a Zeiss Axioplan 2 (Carl Zeiss, Welwyn Garden City, U.K., http://www.zeiss.co.uk) equipped with an AxioCam HRc and the AxioVision software and assembled in Photoshop (Adobe Systems Inc., San Jose, CA, http://www.adobe.com). To examine fibrillar collagen orientation, picrosirius red enhancement of collagen birefringence was performed. Hydrated sections were stained for 1 hour in picrosirius solution (0.1% Direct Red 80 [Sigma] wt/vol in 0.13% picric acid), rinsed in tap water, dehydrated, and mounted in DePeX (VWR International, Ltd., Lutterworth, U.K., http://uk.vwr.com). Enhanced birefringence was assessed under polarized light microscopy. Samples for electron microscopy (e.m.) were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and postfixed in 1% osmium tetroxide. Specimens were en bloc stained with 2% uranyl acetate, dehydrated through a graded acetone series, and embedded in TAAB LV resin. Sections were stained with 0.3% lead citrate in 0.1 M NaOH and viewed on an FEI Tecnai 12 BioTwin transmission electron microscope.

Gene Expression Analysis
RNA was isolated from monolayer cell cultures using TriReagent (Sigma) following the manufacturer's protocol. Three-dimensional pellets or discs were disrupted in TriReagent using a small disposable plastic pestle and an aliquot of Molecular Grinding Resin (G-Biosciences/Genotech, St. Louis, http://www.gbiosciences.com), and RNA was extracted from the TriReagent as before. Total RNA (1 µg) was converted to cDNA using MMLV reverse transcriptase (Promega, Southampton, U.K., http://www.promega.com) and random oligonucleotide primers and was diluted 1:4 with distilled H2O before analysis. Real-time quantitative polymerase chain reaction (PCR) was performed in a DNA Engine Opticon II Continuous Fluorescence Detection System (Bio-Rad) using hot start Taq and SYBR green chemistry (Eurogentec, Southampton, U.K., http://www.eurogentec.be). Primer sequences were taken from previously published work (Table 1) or designed for each gene studied using the Primer Express software (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Primers were obtained from Invitrogen or Eurogentec and optimized for each set. Relative expression levels for each primer set were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase by the 2–{Delta}Ct method [46]. The expression of collagen IIA and IIB isoforms was also assessed by PCR using previously described parameters [13]. Amplification products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.


View this table:
[in this window]
[in a new window]

  		Table 1. Nucleotide primers used for reverse transcription-polymerase chain reaction

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Chondrogenic Differentiation of hMSC in Transwell Cultures
The hMSC (5 x 105 cells) were formed into a shallow multicellular layer approximately 8–9 cells deep on the flexible membrane of a Transwell filter 6.5 mm in diameter (Fig. 1A). When cultured in a chondrogenic medium with TGFβ3 and dexamethasone, the cell layer remained covering the membrane and, during the first 7 days, the DNA content increased eightfold, suggesting approximately three rounds of cell division (Fig. 1B). Beyond 7 days there was a much-reduced rate of proliferation, but the wet mass of the discs increased rapidly to achieve an approximately 20-fold increase in mass by day 14. To assess the performance of the hMSC in the Transwell cultures, they were compared with parallel chondrogenic cultures with the same number of identical cells in identical medium but as cell pellets in a well-established procedure [13, 14, 36]. The contrast was dramatic, as at 14 days the Transwell cultures formed translucent, flexible discs of cartilage of uniform thickness (~0.8 mm), which were more than twice the wet mass of control cell pellets (Fig. 1C). With extended culture to 28 days, the disc thickness showed only a small increase to ~1 mm, but the cartilage disc became more robust and was easily removed intact from the membrane support. Analysis showed that in the discs the proteoglycan content increased linearly over 28 days (Fig. 1D), forming a cartilage matrix that stained strongly and uniformly with Safranin O (Fig. 1A).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. Analysis of human bone marrow stem cells in chondrogenic Transwell culture. (A): Histological distribution of sulfated polyanion in sections of disc cultures at 1, 7, 14, and 28 days of culture revealed by staining with Safranin O/Fast Green. The Transwell membrane can be seen underlying the dense cell layer at day 1 (bar = 100 µm). (B): DNA content of disc cultures over 28 days. (C): Comparison of pellet and disc wet mass after 14 days of culture (*** = p < .001, n = 10). (D): Time-dependent accumulation of GAG relative to DNA content during chondrogenic disc culture. Error bars show mean ± SE. Abbreviation: GAG, glycosaminoglycan.

 
Proteoglycan and Collagen Production in Transwell Cultures
The progress of cartilage matrix assembly in the disc cultures was assessed by determining the proteoglycan content and the cumulative release of proteoglycan into culture medium. Results showed that discs at 14 days accumulated 50% more proteoglycan per cell than the control pellet cultures (Fig. 2A). Furthermore, there was more efficient retention of proteoglycan within the disc cartilage matrix, as 76.9% of the total proteoglycan produced was retained in discs at day 14 versus 65.2% in pellets, and, at day 28, the retention was 75.8% versus 64.3% (Fig. 2B). The total proteoglycan synthesis and the amounts of proteoglycan released into the culture medium were greater in the Transwells. For both discs and pellets, the proteoglycan release per day rose over the first 7 days and then remained relatively constant up to 28 days (Fig. 2C).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		Figure 2. Release kinetics and accumulation of GAG in disc and pellet cultures. (A): Comparison of amounts of GAG accumulated in the matrix of pellet and disc cultures over 14 days (normalized to DNA content). (B): The relative amounts of total GAG produced and retained in the matrix of the constructs compared with that released into the culture medium in discs and in pellets at both 14 and 28 days of culture. Numbers above gray bars indicate percentage of GAG retained for each type of culture. (C): The rate of release of GAG to the culture medium over 28 days of culture. All values are mean ± SE; ** = p < .01, n = 3. Abbreviation: GAG, glycosaminoglycan.

 
The growth in the Transwell culture wet mass was rapid up to 14 days but much slower from days 14 to 28; however, proteoglycan accumulation was linear throughout. Thus, whereas at 14 days the proteoglycan content was 1.5% (wt/wt), by 28 days it had increased to 2.8% (wt/wt) (Table 2). The total collagen content in the Transwell discs also increased 1.8-fold between 14 and 28 days and reached 1.9% of the wet mass (Table 2). The relative amounts of collagens type II and type I were assessed following pepsin digestion of whole samples of discs and pellets. Collagen I is composed of two {alpha}1(I) chains and one {alpha}2(I) chain, whereas collagen II contains three {alpha}1(II) chains. In SDS-PAGE analysis of pepsin digests, the collagen I {alpha}1 and II {alpha}1 chains both migrate together and are separated from the collagen I {alpha}2 chains. Densitometric analysis of the stained gel revealed the ratio of {alpha}1 to {alpha}2 chains and, thus, an estimate of the relative amount of collagen I and II in each sample. The results showed a greater collagen {alpha}1/{alpha}2 ratio of 10.5:1 in Transwells compared with 8.4:1 in pellets, indicating that the collagen in the Transwell disc tissue had a higher collagen II content than in the pellets.


View this table:
[in this window]
[in a new window]

  		Table 2. Comparison of extracellular matrix accumulation in disc cultures with time

 
Localization of Extracellular Matrix Molecules
Immunolocalization was used to assess the morphological distribution of matrix components in Transwell discs, as previous results had shown that chondrogenic pellets were typically rather heterogeneous. At day 14, collagen II was uniformly distributed throughout the disc tissue, whereas in pellet cultures it was less homogeneous, and in the central region it was sparse (Fig. 3A, 3B). Collagen type I was sparse throughout the discs and reflected the high collagen II/I ratio, whereas collagen I in the pellets was distinctly zonal, with strong immunoreactivity at the surface and toward the center of the pellets (Fig. 3C, 3D). Collagen X distribution at day 14 was very weak in Transwells and absent from the margins of the discs, whereas in pellets it was in a zone 250–500 µm from the surface (Fig. 3E, 3F). Aggrecan was uniformly distributed in both disc and pellet cultures (Fig. 3G, 3H). The Transwells thus formed tissue with a much more uniform distribution of matrix components and cells.


Figure 3
View larger version (104K):
[in this window]
[in a new window]

  		Figure 3. Comparison of matrix synthesis and assembly in disc and pellet culture. (A–H): Immunohistochemical localization of specific extracellular matrix (ECM) molecules in disc (A, C, E, G) and pellet (B, D, F, H) cultures at day 14. Distinct positive staining was detected for collagens type II (A, B), type I (C, D), type X (E, F), and aggrecan (G, H). (I, J): Collagen birefringence enhanced with picrosirius red and viewed under polarized light. Bar = 100 µm. (K): Region of disc culture at day 14 viewed by low power electron microscopy showing the extensive ECM between several adjacent cells. Bar = 5 µm.

 
As cartilage matrix is dominated by collagen II fibers, which are typically fine and randomly oriented, whereas collagen I fibers are more coarse and frequently present in more parallel arrays, we investigated collagen orientation in tissue sections by birefringence under polarized light (Fig. 3I, 3J). In the disc cultures, there was very little birefringence with some fine random fiber orientation (Fig. 3I). In contrast, in pellet cultures there was strong and bright orientation aligned with the pellet surface and also some bright fibrils radiating from the pellet center (Fig. 3J). The fine fibrillar collagen structure in disc was also evident in e.m. micrographs, with fibrils approximately 25 nm (±0.16 nm) in diameter embedded in a well-developed extracellular matrix (ECM) and with highest concentration in the pericellular matrix (Fig. 3K).

Increased Expression of Cartilage-Specific Genes in Transwell Cultures
Differences in gene expression between disc and pellet cultures were determined for a range of cartilage macromolecules, transcription factors, other ECM components, and also for genes marking osteogenic and adipogenic differentiation. At 14 days, collagen type II expression was increased by over 20,000-fold in Transwell cultures compared with the starting monolayer hMSC, and this was significantly twofold higher than in pellets (Fig. 4). The collagen type II gene is expressed as two isoforms differentially regulated during development. In the Transwell cultures, the early developmental form, collagen type IIA, was transiently expressed in early chondrogenesis over the first 3 days of culture, and the expression then switched to the more mature IIB isoform (Fig. 5A). There was some reduction in collagen I expression from monolayer into chondrogenic culture, but no difference between Transwells and pellets (Fig. 4). Collagen X expression was increased in both pellet and disc cultures to a similar extent, while the expression of the minor cartilage collagen type XI was higher in the discs with cells from some donors but not others. The pattern of collagen expression thus showed higher collagen II expression and higher ratios of collagen II/I and collagen II/X. It was noticeable that the large increase in expression of collagen type II during chondrogenesis occurred at the same time as the increase in collagen X, and there was no evidence of any sequential order in their expression (Fig. 5B). The pattern of expression of collagen genes was maintained to 28 days in culture (results not shown), which suggested no evidence of further chondrocyte differentiation. The disc cultures also showed greater increased expression of aggrecan, but increases in link protein were not significant (Fig. 4). In keeping with the stronger chondrogenic signal evident in Transwells, there was significantly lower expression of versican, but there was no increased expression of cartilage oligomeric matrix protein (COMP). Matrilin-1, which is expressed by proliferating chondrocytes in the growth plate, was not significantly expressed in either disc or pellet cultures. In contrast, matrilin-3, which is found in articular cartilage, was upregulated in both, although significantly more in Transwells. Decorin and biglycan mRNA levels were also more significantly elevated in the disc cultures, whereas lumican and chondromodulin expressions were not significantly raised. It was interesting that, considering the stronger chondrogenic effect on many matrix genes in the Transwell cultures, the expression of the transcription factors SOX9, L-SOX5, and SOX6 was not higher than in pellet cultures between days 3 and 14 of culture, while matrix production was at a maximum (results not shown).


Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		Figure 4. Gene expression profile of human bone marrow stem cell chondrogenic cultures. The effects of culture format on gene expression in day 14 pellet and disc cultures were analyzed by real-time quantitative reverse transcription-polymerase chain reaction. Total RNA isolated from undifferentiated monolayer and chondrogenic pellet and disc cultures was analyzed for expression of members of the collagen gene family, extracellular matrix components, the transcription factors SOX9, L-SOX5, SOX6, and Cbfa1, and the transcriptional repressor Bapx1. All y-axes represent gene expression levels normalized to glyceraldehyde-3-phosphate dehydrogenase expression for monolayer (unfilled bars), pellet (gray bars), and disc (black bars) samples as indicated. All values are mean ± SE; * = p < .05, ** = p < .01, *** = p < .001, n = 3. Abbreviations: COMP, cartilage oligomeric matrix protein; n.s., not significant.

 


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Figure 5. Temporal expression of collagen types in the initial stages of Transwell disc culture. (A): RNA samples collected from monolayer MSC cultures and at days 0 to 6 of chondrogenic disc culture were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) for collagen type II transcript levels. Inset: levels of the collagen IIA and IIB isoform transcripts assessed by RT-PCR using primers flanking the alternatively spliced IIA exon over the same time period. Molecular mass markers are indicated to the left of the gel in base pairs. (B): Comparison of the regulation of collagen II (filled symbols) and collagen X (open symbols) transcript levels by real-time PCR over days 0 to 6 of culture. Values are expressed as means ± SE; n = 3. Abbreviations: II A, collagen IIA isoform transcript; II B, collagen IIB isoform transcript; bp, base pairs; Col, collagen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, monolayer MSC cultures; mono, monolayer MSC cultures.

 
To assess whether some hMSC in the cultures were following other differentiation pathways, genes expressed distinctively in adipogenic and osteogenic cells were also examined. Lipoprotein lipase, apolipoprotein, and peroxisome proliferator-activated receptor {gamma}2 were not expressed significantly in the disc (or pellet) cultures at 14 days, thus showing no evidence of adipogenesis. For genes associated with osteogenesis, osteopontin showed very low expression in hMSC in monolayer and was upregulated in the chondrogenic cultures, but less so in Transwells than in pellets (~200-fold compared with ~800-fold, p < .05, n = 3) (results not shown), and osteocalcin was not increased in either. The expression of the osteogenic transcription factor Cbfa1 (Runx2) was downregulated during chondrogenesis in both types of culture, and Bapx1 (also called Nkx3.2), which can act as a repressor of Cbfa1, showed upregulation (Fig. 4). There was thus no consistent evidence of adipogenic or osteoblastic differentiation or evidence of chondrocyte further differentiation and hypertrophy in the Transwell disc cultures.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The culture of human bone marrow stem cells in scaffold-free Transwell cultures elicited a more uniform and much more vigorous response to chondrogenic conditions than the well-established pellet form of culture. The major difference may result from the geometry and growth characteristics of the culture. The cells in the Transwell insert formed a shallow flat disc on the permeable support, providing cell-cell contact that other studies have shown to be necessary for efficient chondrogenesis [13, 14], and positioned all cells close to the nutrient supply with easy diffusion access from above and below. The disc cultures generated impressive cartilage-like constructs in only 2 weeks with a 20-fold increase in wet mass and, compared with control pellet cultures, they had increased collagen II and aggrecan expression, increased proteoglycan synthesis, and more efficient incorporation of the proteoglycan into the matrix. Although culture inserts similar to Transwells have been used for chondrogenic tissue formation with mature cultured chondrocytes and with differentiated MSC within hydrogels [47, 48], they have not previously been used for scaffold-free hMSC chondrogenic differentiation.

The cartilage formed in the Transwell cultures had very distinct phases in its development. There was rapid cell proliferation in the first week accompanying chondrogenic differentiation, after which proliferation was largely turned off. Proteoglycan and collagen deposition were strong by day 7 and continued at a similar rate up to day 28, but the increase in wet mass was much less from 14 to 28 days. As the gain in wet mass was limited between days 14 and 28, despite the increase in proteoglycan content, it suggests that the collagen fibril network was well established at day 14, and this restricted further proteoglycan driven swelling of the ECM. This is supported by the electron microscopy data that revealed an extensive fibrillar network after 14 days in culture (Fig. 3K). There was thus a consolidation of the matrix from days 14 to 28 with a strong increase in dry mass but only a modest increase in wet mass. The disc cultures synthesized more total proteoglycan than pellets and more proteoglycan per cell (GAG/DNA). They also retained more of the proteoglycan within the tissue, despite the fact that the 6.5-mm diameter flat disc in the Transwell insert had more than five times the surface area of a 2-mm diameter spherical pellet. This showed that loss of proteoglycan into the medium was not determined by simple diffusion and suggests that the matrix deposited by the cells in the discs was more efficient at retaining proteoglycan and may reflect the acquisition of a more mature cartilaginous matrix than in the pellets.

Analysis of composition showed that, at day 28, the collagen and GAG contents were 1.9% and 2.8% of the disc wet weight, respectively. The scaffold-free cartilage composition generated in this way remains far from that of mature human articular cartilage (~5% GAG and ~25% collagen), but this is achieved over many years. However, the composition was comparable with that of tissue engineered cartilage generated in vitro from young bovine and porcine chondrocytes [49–55], some of which were grown in bioreactors and often for longer times (6–12 weeks). Mechanical conditioning with cyclic hydrostatic pressure has been shown to improve matrix deposition in MSC pellet cultures [56, 57] and longer culture periods, additional added growth factors, and mechanical stimuli may further increase ECM production in the disc cultures.

The Transwell discs expressed more that twice the amount of collagen II mRNA at 14 days compared with the pellet cultures and, although the matrix collagen content as a fraction of the wet mass or normalized to the DNA content was similar, the disc cultures contained a higher proportion of collagen II compared with collagen I. The birefringence study revealed pronounced collagen orientation aligned with the surface in pellets (Fig. 3J), which was absent from disc cultures. This may denote a great difference in the growth characteristics of the two systems due to the geometry of the cultures. The pellet cultures grew as a sphere with a large continuous increase in surface area. It is likely that the tensile forces thereby created at the surface were transmitted to the cells and may have caused a more fibrogenic response resulting in the increased type I collagen production and its alignment in this zone (Fig. 3D). This contrasted with the Transwell cultures, which grew as a shallow disc increasing in thickness. This would involve only a very small change in surface area with growth, which would generate no comparable tensile forces and no fibrogenic stimuli to compromise the chondrogenic expression. We propose that it is the consequence of this that enables the disc cultures to generate a more robust and homogeneous cartilage matrix. We would thus interpret the major advantage of the Transwell culture as being that it favors the increased production and deposition of a cartilage-like matrix, rather than its effect on chondrogenic differentiation per se. This may have important implications for any system growing cartilage in vitro, as it identifies the importance of nutrient supply and absence of tensional loads within the construct. The flat disc geometry of the Transwell appears to maximize the benefits from these factors.

The very distinctive difference at 14 days between disc tissue and pellet tissue was that its structure was much more uniform and stained strongly throughout for collagen type II and aggrecan and with similar cell to matrix ratio and none of the zonal variations found in pellets. Although previous cloning studies have provided evidence that the plastic adherent fraction of bone marrow mononuclear cells contains cells with differing mesenchymal lineage potential [4, 58], disc and pellet cultures formed from the same preparation of hMSC showed that the differences in uniformity of the deposited matrix were most likely caused by the influence of culture conditions, such as the tensile forces generated in the tissue as outlined above, and not by different subpopulations of cells in different regions of the tissue. The more uniform matrix in the discs may be assisted by a more even presentation of differentiation signals to the cells in the early stages of culture with short diffusion distances to both sides of a thin layer of cells. The Transwell cultures clearly provided good mass transport properties while maintaining a critical cell density and clearly indicated that, although cultured articular chondrocytes appear to respond to lower oxygen concentrations in culture by increasing the quantity of ECM production [59, 60], a low oxygen tension does not appear essential for chondrogenesis in hMSC. Indeed, preliminary experiments have shown that culture with lowered oxygen tension (5%) did not improve hMSC chondrogenic differentiation in the Transwell cultures (Murdoch and Hardingham, unpublished observations).

It was interesting that the much stronger matrix production in disc cultures (over days 3–14) was not accompanied by the sustained upregulation of SOX9, SOX6, or L-SOX5. These transcription factors are essential for matrix protein gene expression and the maintenance of the chondrocyte phenotype [61–63]. It therefore appears likely that the expression of these SOX genes in undifferentiated hMSC was already sufficient to support the matrix production observed. Clearly, there are likely to be mechanisms that complement or enhance the activity of SOX9, -5, and -6 transcription factors during chondrogenic differentiation in hMSC and account for the large increase in matrix production and assembly without a change in gene expression.

Early chondrogenic differentiation in the Transwell cultures was accompanied by a transition in the expression of alternatively spliced transcripts, from immature collagen type IIA to the mature collagen IIB (Fig. 5A). This mimics the changes in expression reported in other examples of chondrogenesis during embryonic development, such as in mesodermal condensation to form cartilaginous long bone rudiments, with the expression of the shorter IIB transcript becoming dominant as the full chondrocyte phenotype is established [64]. The expression of collagen type X, in contrast, was not comparable with that found during chondrocyte differentiation in the growth plate, where it accompanies chondrocyte hypertrophy and occurs significantly later than the upregulation of collagen type II expression in prehypertrophic chondrocytes. In the hMSC, the expression of type X collagen increased at the same time as type II collagen (Fig. 5B), and the collagen II and X expression levels remained similar up to 28 days (not shown). We have also observed expression of other markers of the growth plate phenotype, such as Indian hedgehog, in our experiments (typically 8- to 10-fold upregulation in expression at day 14 of chondrogenic culture [Murdoch and Hardingham, unpublished observations]), and expression of type X collagen and alkaline phosphatase has been previously reported in MSC chondrogenesis [13, 14, 16, 65]. However, the expression of Cbfa1 (another factor potentially driving hypertrophy [66]) was not increased in the chondrogenic cultures. Its expression was highest in hMSC in monolayer under nonchondrogenic conditions, and on day 1 of Transwell culture its expression fell by 89%. By day 7 it was at 0.8% of the expression level found in monolayer, and at day 28 it was still only at 2.8% (results not shown). Furthermore, the expression of Bapx1, a repressor of Cbfa1 transcription [67], was increased, which was also consistent with a lack of progression to chondrocyte hypertrophy, and this was further supported by the suppressed expression of matrilin-1, which is typically found in epiphyseal and growth plate cartilage [68], and the increased expression of matrilin-3, an articular cartilage component. Together, these results indicate that there was no clear progression from chondrogenesis to hypertrophy in the cultures. Although the absence of collagen X deposition has been reported in chondrogenic MSC cultures [18], we routinely detect relatively high type X collagen mRNA but only weak matrix immunostaining in the Transwell cultures. Recently published work by Kafienah et al. [65] suggests that collagen X expression can be reduced in scaffold-based hMSC cultures by the inclusion in the culture medium of factors that regulate growth plate development, such as parathyroid hormone-related peptide.

Other methods for producing larger scale cartilaginous constructs with MSCs have typically used natural or synthetic fibrillar scaffolds, such as polyglycolic acid or esterified hyaluronan or hydrogels, such as collagen and alginate [23–35]. Interaction of hMSC with scaffolds involves surface contact, which would tend to mimic monolayer culture and is likely therefore to inhibit differentiation. In contrast, MSCs cultured in hydrogels maintain a rounded cell morphology, which supports chondrogenesis. The Transwell system carries this further by (a) supporting chondrogenesis without any hydrogel, (b) encouraging early proliferation, which gave an eightfold increase in cell number, and (c) providing a strongly chondrogenic environment with tissue growth and deposition not limited by nutrient supply. A limited scale-up has shown that the Transwell cultures generated constructs of 12-mm diameter and 1-mm depth from 1.7 x 106 hMSC in 2–4 weeks and without any integrated biomaterial scaffold (Murdoch and Hardingham, unpublished observations). The technique also provides a more efficient test of chondrogenic differentiation of stem cells, which is more complete and homogeneous than that routinely obtained using pellet or micromass cultures, and would allow a more sensitive test of factors to promote or modulate chondrogenesis and matrix assembly.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Gary Gibson (Henry Ford Hospital, Detroit, MI) for mouse anti-deer collagen X antibody, John Denton (Laboratory and Regenerative Medicine, University of Manchester, U.K.) for help with the polarized light microscopy, and Professor Peter Clegg (University of Liverpool, U.K.) for COMP and link protein primer sets. We acknowledge the support of the Wellcome Trust to the Wellcome Trust Centre for Cell-Matrix Research. This research was supported by a UK Research Council Award to the UK Centre for Tissue Engineering from BBSRC, MRC, and EPSRC.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Ashton BA, Allen TD, Howlett CR et al. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop 1980;151:294–307.[Medline]

  2. Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–1530.[Abstract/Free Full Text]

  3. Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.[CrossRef][Medline]

  4. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

  5. Wakitani S, Sato T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal cells exposed to 5-azacytidine. Muscle Nerve 1995;18:1417–1426.[CrossRef][Medline]

  6. Phinney DG, Kopen G, Issacson RL et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J Cell Biochem 1999;72:570–585.[CrossRef][Medline]

  7. Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.[CrossRef][Medline]

  8. Caddick J, Kingham PJ, Gardiner NJ et al. Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage. Glia 2006;54:840–849.[CrossRef][Medline]

  9. Dowthwaite GP, Bishop JC, Redman SN et al. The surface of articular cartilage contains a progenitor cell population. J Cell Sci 2004;117:889–897.[Abstract/Free Full Text]

  10. Nishimura K, Solchaga LA, Caplan AI et al. Chondroprogenitor cells of synovial tissue. Arthritis Rheum 1999;42:2631–2637.[CrossRef][Medline]

  11. De Bari C, Dell'Accio F, Tylzanowski P et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001;44:1928–1942.[CrossRef][Medline]

  12. Hunziker EB. Articular cartilage repair: Basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 2002;10:432–463.[CrossRef][Medline]

  13. Johnstone B, Hering TM, Caplan AI et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–272.[CrossRef][Medline]

  14. Yoo JU, Barthel TS, Nishimura K et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg 1998;80:1745–1757.[Abstract/Free Full Text]

  15. Derfoul A, Perkins GL, Hall DJ et al. Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal cells by enhancing expression of cartilage extracellular matrix genes. STEM CELLS 2006;24:1487–1495.[Abstract/Free Full Text]

  16. Barry F, Boynton RE, Liu B et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: Differentiation-dependent gene expression of matrix components. Exp Cell Res 2001;268:189–200.[CrossRef][Medline]

  17. Sekiya I, Colter DC, Prockop DJ. BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 2001;284:411–418.[CrossRef][Medline]

  18. Schmitt B, Ringe J, Haupl T et al. BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high density culture. Differentiation 2003;71:567–577.[CrossRef][Medline]

  19. Sekiya I, Larson BL, Vuoristo JT et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res 2005;320:269–276.[CrossRef][Medline]

  20. Longobardi L, O'Rear L, Aakula S et al. Effect of IGF-1 in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling. J Bone Miner Res 2006;21:626–636.[CrossRef][Medline]

  21. Zhou S, Eid K, Glowacki J. Cooperation between TGF-β and Wnt pathways during chondrocyte and adipocyte differentiation of human marrow stromal cells. J Bone Miner Res 2004;19:463–470.[CrossRef][Medline]

  22. Penick KJ, Solchaga LA, Welter JF. High-throughput pellet culture system to assess the chondrogenic potential of mesenchymal stem cells. Biotechniques 2005;39:687–691.[Medline]

  23. Solchaga LA, Dennis JE, Goldberg VM et al. Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage. J Orthop Res 1999;17:205–213.[CrossRef][Medline]

  24. Angele P, Kujat R, Nerlich M et al. Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge. Tissue Eng 1999;5:545–554.[Medline]

  25. Radice M, Brun P, Cortivo R et al. Hyaluronan-based biopolymers as delivery vehicles for bone-marrow-derived mesenchymal progenitors. J Biomed Mater Res 2000;50:101–109.[CrossRef][Medline]

  26. Ponticiello MS, Schinagl RM, Kadiyala S et al. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000;52:246–255.[CrossRef][Medline]

  27. Li WJ, Tuli R, Okafor C et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials 2005;26:599–609.[CrossRef][Medline]

  28. Wang Y, Kim U-J, Blasioli DJ et al. In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells. Biomaterials 2005;26:7082–7094.[CrossRef][Medline]

  29. Solchaga LA, Tognana E, Penick K et al. A rapid seeding technique for the assembly of large cell/scaffold composite constructs. Tissue Eng 2006;12:1851–1863.[CrossRef][Medline]

  30. Caterson EJ, Nesti LJ, Li WJ et al. Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam. J Biomed Mater Res 2001;57:394–403.[CrossRef][Medline]

  31. Diduch DR, Jordan LC, Mierisch CM et al. Marrow stromal cells embedded in alginate for repair of osteochondral defects. Arthroscopy 2000;16:571–577.[Medline]

  32. Williams CG, Kim TK, Taboas A et al. In vitro chondrogenesis of bone-marrow derived mesenchymal stem cells in a photopolymerizing hydrogel. Tissue Eng 2003;9:679–688.[CrossRef][Medline]

  33. Bosnakovski D, Mizuno M, Kim G et al. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: Influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 2006;93:1152–1163.[CrossRef][Medline]

  34. Wakitani S, Imoto K, Yamamoto T et al. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 2002;10:199–206.[CrossRef][Medline]

  35. Kuroda R, Ishida K, Matsumoto T et al. Treatment of a full-thickness articular cartilage defect in the femoral condyle of an athlete with autologous bone-marrow stromal cells. Osteoarthritis Cartilage 2007;15:226–231.[CrossRef][Medline]

  36. Mackay AM, Beck SC, Murphy JM et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 1998;4:415–428.[Medline]

  37. Tew SR, Li Y, Pothacharoen P et al. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage 2005;13:80–89.[CrossRef][Medline]

  38. Farndale RW, Sayers CA, Barrett AJ. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 1982;9:247–248.[Medline]

  39. Bergman I, Loxley R. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Anal Chem 1963;35:1961–1965.

  40. Niyibizi C, Visconti CS, Gibson G et al. Identification and immunolocalization of type X collagen at the ligament-bone interface. Biochem Biophys Res Commun 1996;222:584–589.[CrossRef][Medline]

  41. Gibson G, Lin DL, Francki K et al. Type X collagen is colocalized with a proteoglycan epitope to form distinct morphological structures in bovine growth cartilage. Bone 1996;19:307–315.[Medline]

  42. Ratcliffe A, Hardingham TE. Cartilage proteoglycan binding region and link protein. Radioimmunoassays and the detection of masked determinants in pellets. Biochem J 1983;213:371–378.[Medline]

  43. Martin I, Jakob D, Schafer D et al. Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthritis Cartilage 2001;9:112–118.[CrossRef][Medline]

  44. Li Y, Tew SR, Russell AM et al. Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng 2004;10:575–584.[CrossRef][Medline]

  45. Selvamurugan N, Kwok S, Partridge NC. Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells. J Biol Chem 2004;279:27764–27773.[Abstract/Free Full Text]

  46. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 2001;25:402–408.[CrossRef][Medline]

  47. Masuda K, Sah RL, Hejna MJ et al. A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: The alginate-recovered-chondrocyte (ARC) method. J Orthop Res 2003;21:139–148.[CrossRef][Medline]

  48. Kavalkovich KW, Boynton RE, Murphy JM et al. Chondrogenic differentiation of human mesenchymal stem cells within an alginate layer culture system. In Vitro Cell Dev Biol Anim 2002;38:457–466.[CrossRef][Medline]

  49. Freed LE, Hollander AP, Martin I et al. Chondrogenesis in a cell-polymer bioreactor system. Exp Cell Res 1998;240:58–65.[CrossRef][Medline]

  50. Riesle J, Hollander AP, Langer R et al. Collagen in tissue engineered cartilage: Types, structure and crosslinks. J Cell Biochem 1998;71:313–327.[CrossRef][Medline]

  51. Vunjak-Novakovic G, Martin I, Obradovic B et al. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 1999;17:130–138.[CrossRef][Medline]

  52. Kafienah W, Jakob M, Demarteau O et al. Three dimensional tissue engineering of hyaline cartilage: Comparison of adult nasal and articular chondrocytes. Tissue Eng 2002;8:817–826.[CrossRef][Medline]

  53. Graff RD, Kelley SS, Lee GM. Role of pericellular matrix in development of a mechanically functional neocartilage. Biotechnol Bioeng 2003;82:457–464.[CrossRef][Medline]

  54. Mauck RL, Wang CC, Oswald ES et al. The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading. Osteoarthritis Cartilage 2003;11:879–890.[CrossRef][Medline]

  55. Hu JC, Athanasiou KA. A self-assembling process in articular cartilage tissue engineering. Tissue Eng 2006;12:969–979.[CrossRef][Medline]

  56. Miyanishi K, Trindade MCD, Lindsey DP et al. Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-β3-induced chondrogenesis by adult human mesenchymal stem cells in vitro. Tissue Eng 2006;12:2253–2262.[CrossRef][Medline]

  57. Angele P, Yoo JU, Smith C et al. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J Orthop Res 2003;21:451–457.[CrossRef][Medline]

  58. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–1166.[Abstract]

  59. Hardingham TE, Oldershaw RA, Tew SR. Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat 2006;209:469–480.[CrossRef][Medline]

  60. Murphy CL, Polak JM. Control of human articular chondrocyte differentiation by reduced oxygen tension. J Cell Physiol 2004;199:451–459.[CrossRef][Medline]

  61. Lefebvre V, Li P, de Crombrugge B. A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J 1998;17:5718–5733.[CrossRef][Medline]

  62. Bi W, Deng JM, Zhang Z et al. Sox9 is required for cartilage formation. Nat Genet 1999;22:85–89.[CrossRef][Medline]

  63. Smits P, Li P, Mandel J et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 2001;1:277–290.[CrossRef][Medline]

  64. Zhu Y, Oganesian A, Keene DR et al. Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-β1 and BMP-2. J Cell Biol 1999;144:1069–1080.[Abstract/Free Full Text]

  65. Kafienah W, Mistry S, Dickinson SC et al. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum 2007;56:177–187.[CrossRef][Medline]

  66. Ueta C, Iwamoto M, Kanatani N et al. Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes. J Cell Biol 2001;153:87–99.[Abstract/Free Full Text]

  67. Provot S, Kempf H, Murtaugh LC et al. Nkx3.2/Bapx1 acts as a negative regulator of chondrocyte maturation. Development 2006;133:651–662.[Abstract/Free Full Text]

  68. Klatt AR, Paulsson M, Wagener R. Expression of matrilins during maturation of mouse skeletal tissues. Matrix Biol 2002;21:289–296.[CrossRef][Medline]




This article has been cited by other articles:


Home page
 Stem CellsHome page
R. A. Oldershaw, S. R. Tew, A. M. Russell, K. Meade, R. Hawkins, T. R. McKay, K. R. Brennan, and T. E. Hardingham
Notch Signaling Through Jagged-1 Is Necessary to Initiate Chondrogenesis in Human Bone Marrow Stromal Cells but Must Be Switched off to Complete Chondrogenesis
Stem Cells, March 1, 2008; 26(3): 666 - 674.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
G. M. Hoben, E. J. Koay, and K. A. Athanasiou
Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines
Stem Cells, February 1, 2008; 26(2): 422 - 430.
[Abstract] [Full Text] [PDF]

This Article
Free via Open Access: OA
Right arrow OA Abstract
Right arrow Full Text (PDF)
Right arrowOA All Versions of this Article:
2007-0374v1
25/11/2786    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map