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First published online November 21, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 422 -430
doi:10.1634/stemcells.2007-0641; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines

Gwendolyn M. Hobena,b, Eugene J. Koaya,b, Kyriacos A. Athanasioua

aDepartment of Bioengineering, Rice University, Houston, Texas, USA;
bBaylor College of Medicine, Houston, Texas, USA

Key Words. Fibrocartilage • Human embryonic stem cells • Tissue engineering

Correspondence: Correspondence: Kyriacos A. Athanasiou, Ph.D., P.E., Rice University, Department of Bioengineering, MS-142, P.O. Box 1892, Houston, Texas 77251-1892, USA. Telephone: 713-348-6385; Fax: 713-348-5877; e-mail: athanasiou{at}rice.edu

Received on August 6, 2007; accepted for publication on November 10, 2007.

First published online in STEM CELLS EXPRESS  November 21, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Experimental Procedures
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human embryonic stem cells (hESCs) are an exciting cell source for fibrocartilage engineering. In this study, the effects of differentiation time and cell line, H9 versus BG01V, were examined. Embryoid bodies (EBs) were fibrochondrogenically differentiated for 1, 3, or 6 weeks and then used to engineer tissue constructs that were grown for an additional 4 weeks. Construct matrix was fibrocartilaginous, containing glycosaminoglycans (GAGs) and collagens I, II, and VI. A differentiation time of 3 or 6 weeks produced homogeneous constructs, with matrix composition varying greatly with cell line and differentiation time: from 2.6 to 17.4 µg of GAG per 106 cells and from 22.3 to 238.4 µg of collagen per 106 cells. Differentiation for 1 week resulted in small constructs with poor structural integrity that could not be mechanically tested. The compressive stiffness of the constructs obtained from EBs differentiated for 3 or 6 weeks did not vary significantly as a function of either differentiation time or cell line. In contrast, the tensile properties were markedly greater with the H9 cell line, 1,562–1,940 versus 32–80 kPa in the BG01V constructs. These results demonstrate the dramatic effects of hESC line and differentiation time on the biochemical and functional properties of tissue-engineered constructs and show progress in fibrocartilage tissue engineering with an exciting new cell source.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Experimental Procedures
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Injuries to the fibrocartilages of the body, especially the knee meniscus, most commonly result in disabling arthritis [1, 2]. Tissue engineering of a replacement tissue offers a possible remedy. Most tissue-engineering strategies have used primary cells, but over time, the field has begun shifting toward stem cells. This shift occurred because of a lack of sufficient autologous healthy tissue to provide enough cells for a tissue-engineered construct. Moreover, the goal of taking only a small biopsy of native tissue and expanding those cells to reach the needed number has been confounded by issues of dedifferentiation, low synthetic capacity, and limited expansion [3–6]. These issues are even more pronounced in fibrocartilage compared with hyaline cartilage, as fibrochondrocytes in vitro show inferior matrix production compared with chondrocytes [7, 8]. Toward a goal of tissue engineering fibrocartilages, such as the knee meniscus, temporomandibular joint disc, and intervertebral disc, both adult and embryonic stem cells may have the capacity to overcome these issues, but they also bring their own challenges.

One of the biggest challenges is differentiating the cells. A common treatment in many differentiation studies is the use of serum-free or low-serum "chondrogenic" medium containing insulin, ascorbic acid, and dexamethasone [9–11]. The addition of a transforming growth factor-β (TGF-β) superfamily growth factor has also been commonly used for chondrogenic differentiation. For example, in human embryonic stem cell (hESC) studies, bone morphogenic protein-2 and TGF-β1 have been studied for their efficacy in inducing chondrogenic differentiation [12–16]. An additional component of this differentiation has been the microenvironment for differentiation. This microenvironment can be in terms of differentiating the embryoid bodies (EBs) in suspension, on a two-dimensional surface, or a three-dimensional scaffold such as a hydrogel or polymer scaffold. This microenvironment can also include the presence of other cell types for the purpose of differentiation (Table 1). How long to differentiate the cells prior to using them in a tissue-engineering strategy is another factor that must be considered. Time frames as short as 8 days have been used [12], whereas Khoo et al. [17] found that hESCs spontaneously differentiated down a cartilaginous lineage after 60 days. Although there is a wide array of studies examining each of these components with adult stem cells [18, 19], the field is just beginning with hESCs.


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  		Table 1. Differentiation strategies for chondrogenesis with hESCs

 
In this study, we used chondrogenic medium as the primary differentiation treatment for hESC EBs in suspension. The time for differentiation was varied from 1 to 6 weeks, with the hypothesis that with increasing differentiation time, more fibrocartilaginous matrix would be produced in the EBs, and the cells from these EBs could be used to create biochemically and functionally fibrocartilaginous tissue constructs. To address this hypothesis, the differentiated cells from the EBs were placed in high-density culture as part of a self-assembly process [20], and the resulting constructs were biochemically and functionally characterized (Fig. 1). Two different hESC lines, H9 and BG01V, were compared in this study as an initial examination of the level of variation in cartilaginous lineage differentiation between hESC lines. Such variation certainly has implications for future clinical work, but it also has important considerations for in vitro studies, particularly since the BG01V line has less stringent culture requirements [21, 22].


Figure 1
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  		Figure 1. Study design: Two hESC lines, H9 and BG01V (BG), were cultured in monolayer and then collected as embryoid bodies (EBs) off the mouse embryonic fibroblast feeder layers. The EBs were then cultured in chondrogenic medium with 1% serum for 1, 3, or 6 wks (referred to as the differentiation time) and then dissociated to single cells and self-assembled in agarose molds. The resulting constructs were evaluated following 2 and 4 wks in self-assembly culture, referred to as assembly time. Constructs are referred to in the text according to their cell line and their differentiation time in EB form. Abbreviations: h9, H9 human embryonic stem cells; hESC, human embryonic stem cell; wk, week.

 

   EXPERIMENTAL PROCEDURES
Top
 Abstract
 Introduction
 Experimental Procedures
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
H9 human embryonic stem cells (H9) (WiCell Research Institute, Madison, WI, http://www.wicell.org) were cultured according to the manufacturer's instructions on irradiated Carworth Farms-1 mouse embryonic fibroblasts (MEFs) (Charles River Laboratories, Wilmington, MA, http://www.criver.com). Colonies were passaged using 0.1% type IV collagenase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) every 4–6 days. BG01V hESCs (BG) (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were cultured according to manufacturer's instructions, and they were similarly grown on irradiated MEFs and passaged every 4–6 days. Embryoid bodies (EBs) were formed by incubating the cells in 0.1% dispase for 15–20 minutes and gently pipetting to remove the EBs from the MEF layer. EBs were resuspended in chondrogenic medium containing 1% fetal bovine serum (Gemini, West Sacramento, CA, http://www.gembio.com), Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L-glucose and L-glutamine (Invitrogen), 1% nonessential amino acids, 0.4 mM proline, 50 µg/ml L-ascorbate-2 phosphate, 100 µg/ml sodium pyruvate, 1% insulin, transferring, and selenium + (ITS+) (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), and 100 nM dexamethasone. To facilitate EB formation, the resulting suspension was placed on 2% agarose (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com)-coated 24-well plates for 4 hours. At that time, the suspension was collected into Petri dishes. EBs were grown in Petri dishes with changes of half the medium every 2 days. EB growth was monitored by light microscopy with a stage micrometer on an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com).

Construct Preparation
EBs were collected after 1, 3, and 6 weeks of differentiation time. A portion of the EBs was set aside for analysis, and the remaining EBs were digested to form a cell suspension. First, EBs were washed in DMEM and digested with 0.05% trypsin-EDTA (Sigma-Aldrich) for 1 hour with stirring. Any remaining matrix was then digested with 0.2% type II collagenase (Worthington Biochemical, Lakewood, NJ, http://www.worthington-biochem.com). The collagenase digestion was terminated when undigested material was no longer visible (15 minutes to 1 hour). The cells were then counted, and 5 x 105 cells were seeded into 3-mm 2% agarose wells and allowed to self-assemble into constructs. Medium changes were performed every other day. The resulting constructs were collected after 2 and 4 weeks (post-assembly time). Samples were taken from the constructs for histology/immunochemistry, quantitative biochemistry, mechanical testing, and flow cytometry analysis. Constructs are referred to in the text according to their cell line (H9 or BG) and their differentiation time in EB form.

Histology and Immunohistochemistry
Samples were frozen and sectioned at 12 µm. Hematoxylin and eosin staining was used to examine EB and construct morphology, Safranin O and fast green staining was used to examine glycosaminoglycan (GAG) distribution, and Picrosirius red staining was used to analyze collagen distribution. To determine whether undesired differentiation had occurred, Von Kossa and oil red O stains were performed for evidence of calcification and adipose tissue, respectively. Immunohistochemical analysis was performed by fixing sections in chilled acetone, rehydrating, treating with 3% H2O2 in methanol, and blocking with horse serum. The following primary antibodies were diluted in phosphate-buffered saline (PBS) and applied for 1 hour: 1:300 rabbit anti-human collagen VI polyclonal antibody (pAb) (United States Biological, Swampscott, MA, http://www.usbio.net), 1:500 rabbit anti-human collagen II pAb (Cedar Lane, Hornby, ON, Canada, http://www.cedarlanelabs.com), 1:750 mouse anti-human collagen I (Chondrex, Redmond, WA, http://www.chondrex.com), and 1:150 mouse anti-human {alpha} smooth muscle actin ({alpha}SMA) (Sigma-Aldrich). Visualization using a secondary biotinylated antibody, the avidin biotinylated complex reagent, and 3,3' diaminobenzidine was performed using the Vectastain kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and counterstaining was done with Harris's hematoxylin. Sections of articular cartilage, meniscal fibrocartilage, bone, and skin tissue were run as positive controls.

Quantitative Biochemistry
Samples were lyophilized for 48 hours and digested in 125 µg/ml papain (Sigma-Aldrich) for 18 hours at 60°C. Cell number was determined using Picogreen Cell Proliferation Assay Kit (Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com). A hydroxyproline assay was performed to gauge total collagen using bovine collagen standards (Biocolor, Belfast, U.K., http://www.biocolor.co.uk). Sulfated GAG was measured with the Blyscan GAG Assay Kit (Biocolor). For all three assessments, four to six samples per group were used.

Biomechanics
Samples were cut to a dog-bone shape using biopsy punches (Miltex, York, PA, http://www.miltex.com) and then glued into paper frames that were placed into the grips of a uniaxial materials testing machine (Instron 5565, Instron, Norwood, MA, http://www.instron.com). Strain was applied at 1% of the gauge length per second until failure. Compression testing was done on an indentation apparatus [23]. Each specimen was attached to the sample holder with cyanoacrylate glue and submerged in PBS. The sample surface was positioned perpendicular to the porous indenter tip, and the tip diameter (ranging from 0.7–1.0 mm) was chosen to be <30% of the construct diameter when possible. The specimen was loaded with a tare load of 0.02 N and allowed to reach tare creep equilibrium, defined as deformation <10–6 mm/second or a maximum creep time of 25 minutes. When tare equilibrium was reached, a step load of 0.07 N was applied. Displacement of the sample surface was measured until equilibrium was reached or a maximum creep time of 60 minutes elapsed. The step load was then removed, and the displacement during recovery was recorded until equilibrium was again reached. The intrinsic mechanical properties of the samples were then determined using the linear biphasic theory [24]. For both tensile and indentation testing, five or six samples were used per test for each group.

Statistics
All data were compiled as mean ± SD, and a three-factor analysis of variance was used, accounting for cell type, differentiation time, and time post-self-assembly. If analysis showed a significant difference, a Tukey's post hoc test or Student's t test analysis was performed to compare sample sets, as appropriate. A significance level of 95% and p < .05 was used in all statistical tests performed.


   RESULTS
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Both the H9s and BGs formed EBs that increased in diameter over time from 453 ± 139 µm at 1 week to 833 ± 353 µm at 3 weeks; there was no significant difference between the cell types, so averages for each time point were combined. The EBs did not increase significantly in size from 3 to 6 weeks. Cystic fluid-filled spaces within the EBs were evident throughout their development. As the EBs matured, the intensity of collagen I staining increased, as did staining for collagen VI (Fig. 2). Collagen II staining appeared most intense in the 1- and 3-week EBs. The 3-week EBs for both cell lines displayed all three collagens. Staining appeared in patches suggestive of localized collections of cells producing specific matrix components.


Figure 2
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  		Figure 2. Embryoid bodies prior to self-assembly: H&E shows increasing matrix with increasing differentiation time. In both the H9 and BG EBs, Col I and VI appeared to increase over time, whereas Col II appeared most intense at 3 wks. Embryoid bodies are characterized by heterogeneous shape and size, as well as the presence of cystic spaces. Brown color is indicative of positive staining. Scale bar = 300 µm. Abbreviations: BG, BG01V; Col, collagen; h9, H9 human embryonic stem cells; wk, week.

 
At the end of each differentiation time, the cells were self-assembled in 3-mm molds. After 4 weeks of further culture, the gross characteristics varied greatly (Fig. 3, top). The H9 1-week constructs had an irregular appearance, and constructs were contracted to a diameter of 1.5 ± 0.3 mm. In contrast, the 3-week constructs were smooth and significantly larger at 2.9 ± 0.3 mm in diameter. The BG constructs showed a similar trend, with the 1- and 6-week constructs being severely contracted, whereas the 3-week constructs were 3.3 ± 0.2 mm in diameter. The consistency of the constructs was further examined with hematoxylin and eosin staining (Fig. 3, bottom). The H9 3-week constructs showed uniform matrix formation; the 6-week constructs had a cell-dense appearance with packed nuclei. Whereas the BG 1-week constructs were uniform, the 3-week samples showed numerous small cystic structures throughout the construct matrix. The 6-week constructs had a thick rind of cells (~200 µm thick) with a loose inner matrix, suggesting formation of a central macroscopic cystic space. Picrosirius red staining showed collagen in all of the constructs, and Safranin O staining indicated minimal GAG presence (data not shown). The collagen was further evaluated with immunohistochemistry for collagens I, II, and VI (Fig. 4). In the H9 constructs, collagen I was distributed throughout the construct, and staining increased over time. In contrast, collagen I increased over time for the BG 1-week constructs but appeared to decrease in the 3- and 6-week constructs. In the H9 constructs, collagen II appeared to increase over time in the 1- and 3-week constructs, and these appeared more intense than the 6-week constructs. The staining was less pronounced in the BG constructs and appeared to decrease over time in the 3-week constructs. Similar to collagen II, collagen VI increased over time in the H9 1- and 3-week constructs but appeared to decrease in the 6-week constructs. Staining for collagen VI was very intense in all of the BG constructs. Immunohistochemistry for {alpha}SMA showed an increase in intensity with increasing differentiation time (supplemental online Fig. 7). Staining for calcification or lipid droplets was negative (data not shown).


Figure 3
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  		Figure 3. Constructs after 4 wks of culture post-self-assembly. Top: Gross constructs. All constructs began at 3 mm, and those made from cells differentiated for 3 wks largely retained the initial dimensions or grew, whereas the other groups tended to contract (hash marks indicate millimeters). Bottom: H&E staining of constructs showing construct morphology. Cystic structures are particularly notable in the BG 3-wk constructs. Scale bar = 300 µm. Abbreviations: BG, BG01V; h9, H9 human embryonic stem cells; wk, week.

 


Figure 4
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  		Figure 4. Coll immunohistochemistry. (A): Staining for Coll I shows increasing intensity over time for the H9 1- and 3-wk constructs, whereas the H9 6-wk and BG 3- and 6-wk constructs appear to have less Coll I over time. (B): The H9 1- and 3-wk constructs stained most intensely for Coll II. (C): Coll VI staining appeared to increase over time for all constructs. Brown color is indicative of positive staining. Scale bar = 300 µm. Image shown is a cross-section of a construct including approximately half the total diameter and a lateral edge. Abbreviations: BG, BG01V; Coll, collagen; h9, H9 human embryonic stem cells; wk, week.

 
Biochemical analysis for cells per construct showed that there was significant proliferation in three groups: H9 1 week, BG 3 weeks, and BG 6 weeks (Fig. 5A). Collagen normalized to cells showed that the cell type and the time post-self-assembly were significant factors, with the H9 groups having significantly more collagen, which increased over time, whereas the BGs showed declining collagen over time (Fig. 5B). GAG per cell was not significant for any factors and ranged from 2.6 to 17.4 µg per 106 cells (Fig. 5C). When normalized to dry weight (DW), the self-assembly time was not significant for collagen or GAG content (supplemental online Table 2). Collagen per DW ranged from 15.93% to 22.97% for the H9s and from 8.55% to 9.40% for the BGs. GAG per DW was highest in the H9 1-week constructs at 1.79%, and in the other groups, it ranged from 0.44% to 1.00%.


Figure 5
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  		Figure 5. Quantitative biochemistry. (A): Cells per construct. The H9 1-wk group and the BG 3- and 6-wk groups were notable for significant proliferation over time. (B): Collagen per million cells. Only the cell line was significant with H9s producing greater collagen on a per-cell basis. (C): GAG per million cells showed no significant differences across the groups. *, p < .05. Abbreviations: BG, BG01V; GAG, glycosaminoglycans; h9, H9 human embryonic stem cells; wk, week.

 
Biomechanical testing of the constructs showed that aggregate modulus values ranged from 14 to 42 kPa, and only the BG 6-week constructs were significantly softer than the others, although it is notable that the BG 1-week constructs were too small to be accurately tested (Fig. 6A). The tensile properties differed significantly between the cell types, with the H9s showing an order of magnitude greater tensile modulus (Fig. 6B). Neither the H9 nor BG 1-week constructs could be tested under tension because of their fragility and small dimensions.


Figure 6
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  		Figure 6. Mechanical properties. (A): Aggregate modulus indicating stiffness showing values nearing 50% of native tissue. The BG 1-wk constructs lacked mechanical stability sufficient for handling the sample, and thus, they could not be tested. (B): Tensile modulus. (C): Ultimate tensile strength. The marked superiority of the H9 constructs likely reflects the greater collagen density and homogeneity of the constructs. Neither the BG 1-wk or H9 1-wk constructs could be tested due to their poor mechanical stability. Groups not connected by the same letter are statistically different (p < .05). Abbreviations: BG, BG01V; h9, H9 human embryonic stem cells; wk, week.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Fibrochondrogenic differentiation of hESCs is an exciting and developing field with great potential for clinical application in repairing fibrocartilages, such as the knee meniscus, temporomandibular joint disc, and intervertebral disc. In this study, we examined the effect of differentiation time in EB form on the functional fibrochondrogenic differentiation of two different hESC lines, H9 and BG01V. Using a chondrogenic medium containing 1% serum, dexamethasone, ITS+, and ascorbic acid, EBs were differentiated for 1, 3, or 6 weeks, spanning the differentiation times used in prior studies of cartilaginous differentiation. The resulting cells were then cultured at high density in agarose molds to create neotissue constructs.

The matrix created by the EBs was reflective of the matrix formed in the constructs. In the H9 3-week EBs, collagens I, II, and VI were present, and the constructs showed similar matrix characteristics. In contrast, the 6-week-old EBs had minimal collagen II, and the resulting constructs appeared to contain greater amounts of collagens I and VI. In the BG EBs, both the 1-week and 3-week differentiation periods appeared to produce all three collagens, whereas collagen II dropped off at 6 weeks. Similarly, the resulting constructs contained all three of these matrix components. The presence of collagen VI, recently shown to be particularly important in fibrocartilage development [25–27], is very promising in this study. The loss of collagen II by 6 weeks and the increase in collagen I staining, especially in the BG EBs, is suggestive of a transition toward a more fibroblastic lineage. The contraction of the 6-week constructs from the original 3-mm diameter of the wells and the more intense {alpha}SMA staining in these longer-differentiated constructs further supports this hypothesis. The relevance of {alpha}SMA in fibrocartilage tissue engineering stems from its presence in the native tissue, although its overproduction represents a wound healing or dedifferentiated state [28–31]. These results emphasize the importance of identifying an appropriate window of differentiation prior to moving forward to a tissue-engineering strategy.

The differentiated cells were then gauged for their utility in a tissue-engineering strategy that has been used with primary chondrocytes and fibrochondrocytes [7, 20]. Morphologic characteristics, as well as the matrix composition, translated to the mechanical properties of the constructs. Specifically, since the 1-week differentiated constructs tended to be small and inhomogeneous, they did not handle well for mechanical testing. In addition, as GAG content did not significantly vary across the constructs and GAGs are thought to play a principal role in resisting tissue compression [32], the compressive stiffness was similar for all of the groups. The tensile properties were markedly greater in the H9 constructs, 1,562–1,940 kPa, versus 32–80 kPa in the BG constructs. This difference can likely be attributed to collagen content and organization in the constructs: the BG 3-week constructs had notable cystic structures, whereas the 6-week constructs had a distinct rind devoid of collagen. The biochemical data also suggest that the BGs were in a proliferative state, more than doubling in cell number over time, whereas the H9s showed greater matrix synthesis per cell. In examining the assembly time, the H9s generally showed increasing trends in matrix production over time, whereas the BGs were unchanged or decreased. In looking toward future studies refining and improving the differentiation process, H9 EBs differentiated for 3 weeks prior to construct formation appear to be the most useful model for further work in making functional neofibrocartilage from hESCs.

This work compares favorably with prior work using hESCs toward cartilaginous differentiation, although it is the first to our knowledge to directly address fibrocartilage. A study using H1 and H9 cell lines grown in micromasses for 3 weeks found 1–2.5 µg of GAG per microgram of DNA [13]; in this study, it ranged from 0.34 to 2.4 µg of GAG per microgram of DNA after 4 weeks in self-assembly culture. When BG01V cells were differentiated for 4 weeks in embryoid body form and then self-assembled, collagen and GAG density were similar to that found here [16]. Similarly, a study of BG02 hESCs embedded in hydrogels by Hwang et al. [14] reports 7–20 µg of collagen per microgram of DNA after 3 weeks of culture, whereas we found 2.9–30.9 µg of collagen per microgram of DNA. Overall, the constructs prepared in this study compare favorably to prior work in the cartilage tissue engineering field using different chemical stimuli and microenvironments for differentiation.

Another important component in comparing with previous studies is noting the cell line that was used. In the study by Toh et al. [13], it was found that differences between H1 and H9 cells in chondrogenic differentiation were not significant, and a similar conclusion was reached by Sottile et al. [33] in terms of osteogenic differentiation of the same cell lines. In this study, the cell line was found to be a significant factor in cell proliferation, collagen production, and tensile modulus. Qualitative histological examination also revealed important morphological differences between the constructs resulting from the two cell lines. Work with mouse embryonic stem cells shows similar tendencies, with variation among five different cells lines being as much as 80% in terms of chondrogenic nodule formation [34]. Outside of cartilage-oriented work, other studies with hESC lines also show significant variability in lineage-specific gene expression and differentiation [35–38]. For example, Burridge et al. compared four hESC lines for cardiomyocyte differentiation and found differences ranging from threefold to sixfold [39]. The variability between different cells lines is an important issue in stem cell engineering, with implications in applying differentiation strategies developed with one cell line to other lines. This is of particular importance with regard to cell lines that may have research-related advantages, such as the BG01V cell line. Although karyotypically abnormal, XXY +12 +17, this cell line recovers more easily following cryopreservation and is more robust in culture than the genetically normal hESC lines, which are well known for their culture challenges [21]. Although the H9 cell line has clear advantages in a fibrocartilage tissue-engineering strategy, the BG01V line could be an appropriate system to study chondrogenic differentiation and matrix production but is less appropriate for functional and morphological analysis.

The results of this work with embryonic stem cells also compare favorably to recent work toward tissue-engineered cartilage with adult stem cells. Recently, Murdoch et al. [40] reported creation of cartilaginous neotissue using a scaffoldless approach with mesenchymal stem cells and chondrogenic medium supplemented with a growth factor, TGF-β3. The wet-weight percentages of GAGs and collagen were 2.8% and 1.9%, respectively, compared with a range of 0.15%–1.21% GAG/wet weight and 0.89%–5.78% collagen/wet weight for the constructs created in this study with hESCs. Toward more fibrocartilaginous tissue, several groups have examined transplanting mesenchymal stem cells in various scaffolds or carriers into fibrocartilage defects in vivo [41–44]. Results of these studies have varied from no evidence of benefit of the adult stem cells to formation of fibrous matrix and filling of the defect. There has also been in vitro work with adipose-derived adult stem cells, as well, toward fibrocartilage engineering [45]. Although adult stem cells are promising, there are concerns with the expansion capacity [46], decreasing plasticity with aging [47, 48], specific serum dependence [49, 50], and relative scarcity [51, 52]. There are significant obstacles in using embryonic stem cells, but they do offer advantages, including expansive proliferative potential [53] and pluripotency [54]. For these reasons, we believe it is imperative that the fibrochondrogenic capacity of both types of stem cells be fully explored.

Further work with this model toward fibrocartilage engineering will likely focus on increasing GAG synthesis, as well as modulating specific collagen formation. The characteristics of these constructs are overwhelmingly fibrocartilaginous given the strong collagen I and VI presence. Matrix synthesis, when normalized to dry weight, shows GAG synthesis on the low end of the 1%–7% GAG per DW range found in native meniscal tissue, whereas collagen content is much lower than in native tissue, which averages 80% [55, 56]. Functional evaluation via mechanical testing revealed mechanically stable constructs when using cells differentiated for 3 weeks or more. The compressive stiffness was within 47% of the lower end of the range in human meniscal tissue, whereas the tensile modulus is an order of magnitude lower [32, 57]. The modular approach of this work, incorporating a differentiation step and a self-assembly step, paves the way for future work refining the process at both steps. Having identified a critical 3-week window for differentiation during which the important fibrocartilage proteins develop, future studies may focus on refining the differentiation protocol, as well as using different stimuli during the self-assembled phase to enhance the constructs. One way in which the differentiation step may be modified is to introduce a step to purify the differentiated population using cell surface markers or even a Percoll gradient [58, 59]. In the self-assembly stage, growth factor addition may be a powerful stimulus toward collagen and GAG production, as no growth factors were used in this work. For example, TGF-β1 and insulin-like growth factor-1 have a strong history in enhancing matrix production in fibrocartilage constructs [60–62]. Mechanical stimulation may also be a successful strategy, as has been demonstrated by work with adult stem cells [63]. By enhancing GAG synthesis with further chemical modification or mechanical stimulation, it may be possible to enhance the compressive strength of these constructs, and furthermore, increased collagen production will contribute to tensile strength.

In conclusion, this works demonstrates a significant stride forward in examining the use of hESCs for fibrocartilage tissue engineering. Both the differentiation time and the cell line used play important roles in the success of a tissue-engineering strategy.


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


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This study was supported by National Institute of Arthritis & Musculoskeletal & Skin Diseases R01 AR 47839-2, an unrestricted fund from Rice University, the National Science Foundation-Integrative Graduate Education and Research Traineeship (to E.J.K.), and the Hertz Foundation (to G.M.H.). We thank Prof. Thomas Zwaka of Baylor College of Medicine for helpful discussions.


   REFERENCES
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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