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Keywords:

  • Embryonic stem cells;
  • Mesenchymal stem cells;
  • Hydrogel;
  • Biomechanics;
  • Chondrogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

Cells in the musculoskeletal system can respond to mechanical stimuli, supporting tissue homeostasis and remodeling. Recent studies have suggested that mechanical stimulation also influences the differentiation of MSCs, whereas the effect on embryonic cells is still largely unknown. In this study, we evaluated the influence of dynamic mechanical compression on chondrogenesis of bone marrow-derived MSCs and embryonic stem cell-derived (human embryoid body-derived [hEBd]) cells encapsulated in hydrogels and cultured with or without transforming growth factor β-1 (TGF-β1). Cells were cultured in hydrogels for up to 3 weeks and exposed daily to compression for 1, 2, 2.5, and 4 hours in a bioreactor. When MSCs were cultured, mechanical stimulation quantitatively increased gene expression of cartilage-related markers, Sox-9, type II collagen, and aggrecan independently from the presence of TGF-β1. Extracellular matrix secretion into the hydrogels was also enhanced. When hEBd cells were cultured without TGF-β1, mechanical compression inhibited their differentiation as determined by significant downregulation of cartilage-specific genes. However, after initiation of chondrogenic differentiation by administration of TGF-β1, the hEBd cells quantitatively increased expression of cartilage-specific genes when exposed to mechanical compression, similar to the bone marrow-derived MSCs. Therefore, when appropriately directed into the chondrogenic lineage, mechanical stimulation is beneficial for further differentiation of stem cell tissue engineered constructs.

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


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

There are a number of cell types in the body that can respond to mechanical stimuli and exhibit mechanoresponsive behavior. Mechanoresponsive cell types include chondrocytes in cartilage, osteoblasts in bone, and vascular cells including smooth muscle and endothelial cells. The process of mechanotransduction (transfer of mechanical stimuli to a cell) results in altered gene expression, cell morphology, and extracellular matrix (ECM) production [1, 2]. In the case of cartilage, mechanical signals can significantly modify cartilage structure and are important to cartilage tissue homeostasis [3, 4]. For example, dynamic mechanical compression of chondrocyte-seeded biomaterial scaffolds in vitro increases the production of ECM and improves tissue organization as well as the resulting tissue mechanical properties [5, [6], [7], [8], [9], [10]11].

Mesenchymal stem cells, derived primarily from the bone marrow, are capable of differentiating into mesenchymal tissues including cartilage, bone, tendon, ligament, muscle, and adipose [12, 13]. MSCs differentiate into the chondrogenic lineage when cultured in a three-dimensional (3D) environment with the appropriate growth factors such as transforming growth factor beta (TGF-β) [14, [15], [16]17]. Mechanical stimulation enhances MSC chondrogenesis, resulting in greater proteoglycan and collagen contents [18, [19]20], upregulation of aggrecan, Sox-9, and type II collagen gene expressions [18, 21, [22]23], and greater compressive strengths [19]. These studies demonstrated that not only did mechanical stimulation promote tissue formation by chondrocytes, but it also enhanced chondrogenic differentiation of stem cells [24].

Although MSCs continue to demonstrate promise as a cell source in tissue engineering applications, they have some limitations in their proliferative and differentiation potential during ex vivo expansion [25]. Alternatively, embryonic stem cells may provide an unlimited source of cells for research and therapeutic applications due to their capacity to self renew and differentiate into all three germ layers [26]. We have recently derived cells from human embryonic stem cells that exhibit similar morphology, cell surface markers, gene expression profiles, and mesenchymal differentiation capacity to MSCs [27]. Although enhanced adult and embryonic progenitor cell differentiation in the presence of different forms of mechanical stimulation has been observed [28, 29], the effect of mechanical compression on the chondrogenic differentiation of MSCs encapsulated in hydrogels has not been studied. Furthermore, the effect of mechanical compression on embryonic stem cells and chondrogenesis has not been tested in any form. Hydrogels are appealing 3D systems for tissue engineering as they can mimic the ECM network that cells form and are embedded into during tissue development, and they can eventually be implanted with minimally invasive surgeries [30, 31]. It might be that mechanical stimulation in these 3D constructs has a similar beneficial effect as seen in bidimensional cultures or in other 3D matrices for MSCs. It might also be possible that the mechanical stimulation would result in a different response of adult and embryonic progenitor cells due to the different potencies (multiple vs. total) of these lineages. From a regenerative point of view, it is also relevant for improved tissue engineered constructs to evaluate whether mechanical and chemical (e.g., administration of growth factors) cues have a synergistic or antagonistic effect. Therefore, the purpose of this study was to evaluate the response of adult and embryonic mesenchymal progenitor cells to mechanical compression after encapsulation in hydrogels and culture in chondrogenic conditions with and without growth factor (TGF-β1) conditioning.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

MSC Isolation and Culture

MSCs were isolated from the bone marrow retrieved from the iliac crest of castrated male goats approximately 3 years of age. The bone marrow aspirates were processed within 4 hours of harvest and washed with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The cell suspension was placed in MSC growth medium (Cambrex, Walkersville, MD, http://www.cambrex.com) at a density of 1 × 105 mononuclear cells per cm2. Adhered cells were then collected using 0.025% Trypsin/EDTA (Cambrex) and plated at a density of 5 × 103 cells per cm2.

Derivation of Mesenchymal Progenitors from Human ESCs

The BG01 and BG02 human embryonic stem cell lines (BresaGen, Thebarton, SA, Australia) were cultured as previously described [7]. Briefly, ESCs were cultured on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts in culture medium (80% Dulbecco's modified Eagle medium [DMEM, Invitrogen], 20% fetal bovine serum [FBS], 1% nonessential amino acid, 1 mM l-glutamine, and 0.1 mM β-mercaptoethanol). Colonies were then removed from the dish using splitting medium (1 mg/ml collagenase IV [Invitrogen] in DMEM) and the dissociated cells were transferred to Petri dishes at a density of 10,000 cells per cm2. Embryoid bodies were formed and allowed to grow in suspension for 5 days before being transferred to tissue culture plates. EBs were cultured for an additional 10 days until outgrowth from the EBs occurred as previously described [27]. The cells were harvested using 0.05% Trypsin (Invitrogen), filtered to remove cell clumps, and expanded in medium containing 90% DMEM, 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. After the first passage, the human embryoid body-derived cells (hEBd cells) were expanded at a density of 5 × 103 cells per cm2.

Cell Encapsulation in Hydrogels

Cells were encapsulated in poly(ethylene glycol)-diacrylate hydrogels (PEGDA). MSCs (passage 3 or 4) were suspended in 10% wt/vol PEGDA (SunBio, Orinda, CA, http://www.sunbio.com) solution in sterile PBS with 100 U/ml penicillin and 100 μl/ml streptomycin (Gibco, Grand Island, NY, http://www.invitrogen.com). Photoinitiator Irgacure 2959 (Ciba Specialty Chemicals, Tarrytown, NY, http://www.cibasc.com) was used to initiate the reaction at 0.05% (wt/vol) final concentration. The photoinitiating and encapsulation processes have been previously determined to be biocompatible [32]. MSCs were resuspended in 95 μl of polymer solution at a concentration of 20 × 106 cells per milliliter. The cell-polymer solution was added to a cylindrical silicone-tubing mold with an internal diameter of 4.75 mm and a construct height of 5 mm. The tubing was attached to the glass microscope slide using silicon lubricant. Prepolymer (macromer) was exposed to UV light (λ = 365 nm) for 5 minutes to induce gelation.

hEBd cells (passage 3 or 4) were encapsulated in tyrosine-arginine-glycine-aspartate-serine (YRGDS)-modified hydrogels (YRGDS-PEG-acrylate) as previously described [33, 34]. Acryl-poly(ethylene glycol) N-hydroxysuccinimide (Acry-PEG-NH2, 70 mg; SunBio) was reacted with 5 mg of YRGDS and 2 ml of 50 mM Tris (pH 8.2) at room temperature for 2.5 hours. The solution was lyophilized. YRGDS-NH-PEG-Acryl and PEGDA were mixed in sterile PBS to create a 2.5 mM RGD polymer solution and added to hEBd cells to make a density of 20 × 106 cells per milliliter. YRGDS-PEGDA cell solutions were photopolymerized similar to the PEGDA hydrogels.

Constructs were removed from the molds and cultured at 37°C, 5% carbon dioxide in chondrogenic medium (DMEM, 100 nM dexamethasone [Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com], 40 μg/ml proline [Sigma], 50 μg/ml ascorbic acid, 1% sodium pyruvate [Invitrogen], penicillin/streptomycin, and insulin-transferrin-selenium acid premix [BD Biosciences, San Diego, http://www.bdbiosciences.com]). The scaffolds were cultured either in chondrogenic medium alone or supplemented with TGF-β1 (10 ng/ml; Research Diagnostics, Concord, MA, http://www.researchd.com) prior to and during mechanical stimulation.

Mechanical Loading with Compression

A custom-built bioreactor system was designed to apply unconfined, cyclic compressive strain to cell-seeded scaffolds [35]. The system was driven by air pressure (constant at 0.135 MPa) regulated by a digital control box. The valves, regulated by a timer, open and close to control air flow to the piston, producing the cyclic motion of the piston. The stainless steel compression rods translated relative to fixed cages, transmitting cyclic motion to the cells through direct contact with the hydrogel (Fig. 1A, 1D). In all experiments, the bioreactor induced a strain of 10% of the hydrogel constructs at 1 Hz frequency. Each of six cylindrical-shaped cages held one construct and had a 5.5-mm inner diameter and a height of 5.5 mm (Fig. 1B). These cages contained small holes for medium exchange (Fig. 1D).

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Figure Figure 1.. Mechanical compression bioreactor. (A): Interior of stimulation chamber, including stainless steel rods and plastic stopper. (B): Base of stimulation chamber, which houses cell-seeded constructs in six cages. (C): Petri dish of silicon tubing molds for hydrogel formation and cell encapsulation. (D): Assembled bioreactor includes metal housing, piston, and stimulation chamber. Control box regulates magnitude and frequency of the air pressure. (D1): Air flow-in from pressurized air supply. (D2): Timer that controls air valve. (D3): Indicator that controls the magnitude of air pressure. (D4): Air outlet tube that drives the piston.

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Cells were encapsulated in hydrogels and incubated in static culture before mechanical stimulation was applied. After stimulation, the constructs were removed from the bioreactor and cultured in 24-well plates until the next stimulation period or were harvested immediately for analysis. With or without TGF-β1, the constructs containing MSCs were subjected to mechanical compression for 1, 2, 2.5, or 4 hours per day for 1, 2, and 3 weeks (Fig. 2A). In the case of hEBd cells, day 0 was taken 4 days after encapsulation to ensure that cells attached to the RGD domains in the gels, since otherwise they would not start chondrogenesis as previously shown [27]. Mechanical stimulation was performed for 30 minutes and 1 hour without TGF-β1 or for 1 and 2 hours with TGF-β1 administration. Compression was applied at intervals of 1, 2, and 3 weeks. Different stimulation times were evaluated to determine the optimal mechanical conditions for chondrogenesis.

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Figure Figure 2.. MSC mechanical stimulation in hydrogels. (A): Experiment design and application protocol for mechanical stimulation. (B): qRT-PCR analysis of Sox-9 and aggrecan gene expression normalized to β-actin for MSCs mechanically stimulated for 1 or 2 hours compared with the nonstimulated control (n = 3). (C): Live-Dead assay images immediately after encapsulation, after 24 hours, and 6 days in nonstimulated (C) or mechanically stimulated conditions (10×). No transforming growth factor-β1 conditioning was applied (∗, p < .05). Abbreviations: C, control; hr, hour; MS, mechanically stimulated; PEGDA, poly(ethylene glycol)-diacrylate; RT/qRT-PCR, real-time quantitative reverse transcription–polymerase chain reaction.

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Cell Viability

Cell viability was monitored using the Live-Dead Viability Kit (Invitrogen). Thin slices of constructs were incubated in 1 ml of Live-Dead solution composed of 0.5 μl of calcein AM and 4 μl of ethidium homodimer-1 at 37°C for 30 minutes.

Histology

Constructs were fixed in 10% formaldehyde for 48 hours and stored in 70% ethanol until processing. The constructs were dehydrated in a sequential series of ethanol aqueous solution, embedded in paraffin, and sliced into 5-μm sections. Safranin O and fast green staining was used for specific glycosaminoglycan (GAG) detection.

Biochemical Analysis

After harvest, the wet and dry weights (after 48 hours of lyophilization) of the constructs were measured. Using 1 ml of papainase (papain [type II, 19 U/mg; Worthington Biochemical, Lakewood, NJ, http://www.worthington-biochem.com], 100 mM phosphate buffer, 10 mM cysteine EDTA, pH 6.3), constructs were crushed and digested for 16 hours at 60°C. The DNA content was then measured using Hoechst 33258 dye (Sigma) read by a fluorometer. Calf thymus DNA (Invitrogen) was diluted in 10× TNE buffer as standard [36]. The GAG was assayed with dimethylmethylene blue (Sigma), and chondroitin sulfate (Sigma) was used as a standard to quantify the amount of GAG as previously described [37]. DNA and GAG content was normalized by the wet weight of hydrogel constructs.

Reverse Transcription-Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction

Total RNA was extracted from constructs with 1 ml of TRIzol reagent (Invitrogen) according to manufacturer's protocol. Double-stranded cDNA was created from 1 μg of RNA using the SuperScript First-Strand System (Invitrogen). Reverse transcription-polymerase chain reactions (PCRs) were composed of 1 μl of cDNA per 25-μl volume and Taq DNA Polymerase (Invitrogen). PCR was performed on type II collagen, aggrecan, and Sox-9. Primers (Table 1) were synthesized by MWG Biotech (Ebersberg, Germany, http://www.mwg-biotech.com). PCR was completed using the following cycles: (a) 95°C for 4 minutes and (b) 35 cycles of 30-second denaturation at 95°C, 30-second annealing at the primer-specific temperature, and a 1-minute elongation at 72°C. PCR products were separated by electrophoresis at 100 V on a 2% agarose gel in Tris-acetate-EDTA buffer and then visualized with ethidium bromide staining. Real-time–PCR (RT-PCR) reactions were performed using SYBR Green PCR Master Mix and monitored in the ABI Prism 7700 Sequence Detection System (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). One microliter of cDNA was used per total reaction volume of 25 μl. For PCR and RT-PCR, genes of interest were analyzed and normalized with β-actin. The levels of expression of each target gene were calculated as −2ΔΔCT as previously described [27, 38].

Table Table 1.. Sequences of primers and settings used in reverse transcription-polymerase chain reaction and real-time polymerase chain reaction
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Statistical Analysis

Values in this study are reported as mean and standard deviation. Statistical analysis was performed using one-factor analysis of variance and a Tukey post hoc assessment of the differences between samples, where the confidence level was set to .05 for statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

MSC Response to Mechanical Compression

Differentiation and production of cartilage-related markers increased in MSCs exposed to dynamic compression. MSC-seeded hydrogels cultured without TGF-β1 were exposed to one cycle of mechanical compression for 1 or 2 hours on the 3rd day after encapsulation and harvested immediately. Live-Dead assay showed cell viability was maintained after encapsulation and during culture (Fig. 2C). RT-PCR demonstrated significant increases in Sox-9 and aggrecan gene expression in MSCs after mechanical stimulation (Fig. 2B). After 1 hour of compression, encapsulated MSCs upregulated Sox-9 and aggrecan gene expressions 1.7- and 3.8-fold, respectively, compared with control hydrogels that were not exposed to mechanical stimulation. Applying compression to the MSC hydrogels for 1 or 2 hours produced similar expressions of Sox-9 and aggrecan.

To determine the effect of more prolonged exposure to mechanical compression and optimal differentiation conditions, MSCs in hydrogels were cultured with TGF-β1 and dynamically compressed for 2.5 hours per day for 6 and 14 days and 4 hours per day for 6, 14, and 21 days. The nonstimulated controls consisted of MSCs encapsulated in hydrogels and statically cultured in chondrogenic medium supplemented with TGF-β1. RT-PCR analysis performed on mechanically stimulated hydrogels after 14 days demonstrated significant increases in chondrogenic gene expression (Fig. 3A). Sox-9 expression increased 1.2-fold after 2.5 hours per day of mechanical compression. However, there was no additional increase for groups stimulated for 4 hours per day compared with nonstimulated controls. Aggrecan gene expression increased 1.4- and 2.5-fold after 2.5 and 4 hours per day of mechanical stimulation, respectively. Quantified type II collagen gene expression increased by a factor of 2.1 after 2.5 hours per day of mechanical stimulation.

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Figure Figure 3.. Real-time polymerase chain reaction quantification of Sox-9, aggrecan, and collagen II gene expression for MSCs treated with transforming growth factor-β1. (A): Sox-9, aggrecan, and collagen II gene expression of MSCs stimulated for 2.5 and 4 hours a day for 14 days compared with nonstimulated control (n = 3). (B): Biochemical analysis of DNA and GAG contents normalized to the construct wet weight after 2.5 hours and 4 hours of compression (n = 3, 4). (C): Safranin O staining of mechanically stimulated and control hydrogels. Abbreviations: C, control; GAG, glycosaminoglycan; h, hours; hr, hours; MS, mechanically stimulated; WW, wet weight.

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ECM accumulation in the hydrogels also increased with exposure to mechanical compression. GAG content increased 1.3-fold in hydrogels that were stimulated 2.5 hours per day for 2 weeks. Safranin O staining on hydrogels stimulated for 4 hours confirmed GAG production. GAG was localized pericellularly, while the staining intensity increased over culture time (Fig. 3C). Similarly, GAG content measured in groups stimulated for 4 hours per day increased as compared with nonstimulated controls. In this case, GAG accumulation steadily increased over the time of stimulation (Fig. 3B). Specifically, an increased mechanical stimulation time from 2.5 to 4 hours per day resulted in a further increase in GAG production from 1.3-fold to 2.0-fold. These results were consistent with increased aggrecan gene expression after 14 days of mechanical stimulation. Furthermore, DNA assay showed a negligible difference in cell proliferation between the mechanically stimulated groups and nonstimulated controls over the culture period (Fig. 3B).

hEBd Cell Response to Mechanical Compression in the Absence of TGF-β1

hEBd cells were cultured in YRGDS-PEGDA hydrogels for 7 days prior to the application of mechanical stimulation to ensure cell adhesion to the hydrogel RGD domains and chondrogenesis initiation as previously shown [27]. hEBd cells isolated from human embryonic stem cells, encapsulated in hydrogels, and cultured without TGF-β1 responded to mechanical compression by downregulating chondrogenic genes Sox-9, aggrecan, and type II collagen. Sox-9 gene expression of hEBd cells decreased after 1 hour of mechanical compression per day for 1 and 3 days. In addition, a downregulation of aggrecan and type II collagen gene expression was observed after 1 hour of compression for 3 and 6 days (Fig. 4B). Viability studies performed on hEBd cells indicated similar cell survival after 2 hours of mechanical compression as compared with nonstimulated controls (Fig. 4C).

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Figure Figure 4.. Human embryoid body-derived cell mechanical stimulation in hydrogels. (A): Protocol for isolation and culture of human embryoid body-derived cells. (B): Polymerase chain reaction on human embryoid body-derived (hEBd) cell constructs. All groups were cultured for 7 days in chondrogenic medium without transforming growth factor-β1 before MS was applied. On D1, two groups were stimulated for 30 minutes and 1 hour, respectively, and harvested immediately. Mechanically stimulated groups on D3 and D6 were stimulated for 1 hour a day for 3 and 6 days, respectively. Gene expression analysis of β-actin (housekeeping), Sox-9, aggrecan, and collagen II is shown (n = 4). (C): Live-Dead analysis of hEBd cells encapsulated in poly(ethylene glycol)-diacrylate in nonstimulated conditions. (D): Live-Dead analysis of hEBd cell constructs stimulated for 2 hours and harvested immediately (10×). Abbreviations: 2-D, two dimensional; 3D, day 3; 6D, day 6; D1, day 1; min, minute; MS, mechanical stimulation.

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TGF-β1 Conditioning Changes Mechanical Response of hEBd Cells

Conditioning hEBd cells in chondrogenic medium with TGF-β1 altered the mechanoresponsive behavior of the cells. Cell seeded constructs were cultured in TGF-β1 supplemented chondrogenic medium for 1, 2, and 3 weeks postencapsulation before mechanical stimulation was applied (Fig. 5A). Exposure to TGF-β1 induced chondrogenesis in hEBd cells according to the gene expression patterns over 3 weeks of culture (Fig. 5C, 5D). Chondrogenic gene expression was evaluated after one application of mechanical compression for 1 or 2 hours. After 2 weeks of conditioning, mechanical compression enhanced hEBd cell chondrogenic differentiation similar to MSCs.

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Figure Figure 5.. Human embryoid body-derived cell mechanical stimulation in growth factor conditioned hydrogels. (A): Schematic of conditioning experiment. (B): Real-time–polymerase chain reaction (RT-PCR) quantification of Sox-9 gene expression for 1, 2, and 3 weeks of transforming growth factor (TGF)-β1 conditioning. Nonstimulated control compared with 1 or 2 hours of mechanical stimulation. (C): RT-PCR quantification of aggrecan gene expression for 1, 2, and 3 weeks of TGF-β1 conditioning. Nonstimulated control compared with 1 and 2 hours of mechanical stimulation. (D): RT-PCR quantification of collagen II gene expression for 1, 2, and 3 weeks of TGF-β1 conditioning. Nonstimulated control compared with 1 and 2 hours of mechanical stimulation. The normalized fold increase is relative to β-actin. (E): Safranin O staining showing no glycosaminoglycan (GAG) accumulation after 1 week (10×). (F): Safranin O staining depicting GAG accumulation after 3 weeks (×10; inset picture ×40). Four samples per group were used for analysis. Day 0 control was harvested 4 days postencapsulation to ensure human embryoid body-derived cell attachment to the RGD domains as a prerequisite for chondrogenesis initiation [27]. Abbreviations: h, hour(s); MS, mechanical stimulation.

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RT-PCR was used to quantify Sox-9, aggrecan, and type II collagen gene expression. After 1 week of conditioning, no change was observed between the stimulated and nonstimulated groups for Sox-9, aggrecan, or type II collagen gene expression. However, after 2 weeks of conditioning, Sox-9 expression increased 3.6-fold after 2 hours of mechanical stimulation compared with the nonstimulated control (Fig. 5B). There was no significant difference in Sox-9 expression between cells cultured with TGF-β1 for 2 or 3 weeks. Aggrecan gene expression increased 3.5- and 3.0-fold after hEBd cell constructs were exposed to 2 hours of compression after 2 and 3 weeks of conditioning in TGF-β1, respectively (Fig. 5C). Type II collagen gene expression demonstrated a similar trend after 2 or 3 weeks of conditioning in TGF-β1. The fold increase of type II collagen expression after 2 hours of stimulation was 2.9 and 2.5 for 2 and 3 weeks as compared with nonstimulated hydrogels, respectively (Fig. 5D). Strikingly, the increase in aggrecan and collagen type II was over one and two orders of magnitude, respectively, with respect to β-actin. This implies a higher sensibility of hEBd cells to mechanical stimulation than MSCs. ECM formation in hEBd cell encapsulated constructs was also confirmed by histology (Fig. 5E, 5F), as shown by positive staining for GAG accumulation after 3 weeks of culture.

These findings reveal a mechanoresponsive behavior of hEBd cells that was not present without TGF-β1 conditioning. These trends were more consistent with the gene expression patterns observed in mechanically stimulated MSCs. This evidence suggests that TGF-β1 and differentiation conditions modified the response of hEBd cells to their mechanical environment.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

In articular cartilage, chondrocytes respond to mechanical compression and remodel their extracellular matrix, ultimately changing the composition, structure, and biomechanical properties of the tissue. An appropriate loading regime is necessary to balance ECM synthesis and degradation [39]. Examining the effect of mechanical stimuli on the chondrogenic differentiation of stem cells is important in the quest to create functional cartilage tissue and understand stem cell biology. This study investigated the effect of cyclic mechanical compression using 10% strain deformation at 1 Hz frequency on MSC chondrogenic differentiation. In addition, the mechanoresponsive behavior of a novel embryonic stem cell-derived mesenchymal progenitor population was established. No lift-off phenomena were observed during mechanical stimulation. PEGDA gels have been previously characterized as viscoelastic materials with a very minimal viscous response [40]. Therefore, since 10% strain deformation is typically within the elastic region of a polymer mechanical response, elastic deformation can be assumed during the applied cyclic stimulation, and a complete recovery of the hydrogels can be expected every cycle as also previously found [35].

MSCs increased chondrogenic gene expression, specifically Sox-9 and aggrecan, after the application of mechanical simulation in the absence of TGF-β1. In the presence of TGF-β1, mechanical stimulation increased Sox-9, aggrecan, and type II collagen gene expression and matrix production after 14 days of exposure. The increase in chondrogenic differentiation and synergistic effects of mechanical stimulation and TGF-β1 are consistent with other recent studies. Huang et al. demonstrated that rabbit MSC-seeded agarose gels respond to deformation (4 hours per day of 10% strain at 1 Hz frequency) by increasing aggrecan and type II collagen gene expression with or without TGF-β [4]. Similar trends were observed in pellet studies performed by Miyanishi et al., where increases in Sox-9, type II collagen, and aggrecan gene expression after 14 days of 4-hours-per-day stimulation were observed (10 MPa intermittent hydrostatic pressure at 1 Hz frequency) [20, 23]. Huang et al. demonstrated an increase in TGF-β1 receptor gene expression of rabbit MSCs after 1 hour of stimulation, which contributes to a greater affinity to TGF-β1 [22]. This may have led to the upregulation of chondrogenic gene expression in mechanically stimulated groups and corroborates the synergistic effect of chemical and mechanical cues presented to adult and embryonic stem cells on their chondrogenic differentiation in this study. Mauck et al. demonstrated similar increases in GAG accumulation after 3 hours per day of stimulation (1 Hz and 10% strain) for 5 days with a total culture time of 14 and 28 days [17]. In the current study, the increase in GAG content was larger for constructs stimulated for 4 hours as opposed to 2.5 hours, specifically at day 14. This suggests that increases in exposure to mechanical compression augment aggrecan gene expression and, thus, GAG accumulation. The upregulation of chondrogenic gene expression and the increase in GAG accumulation may be attributed to the presence of mechanically sensitive surface receptors on MSCs. Surface integrins found on mature chondrocytes contribute to increases in aggrecan and MMP3 gene expression [41]. Specifically α5β1 integrin, commonly associated with chondrocyte mechanotransduction, might be responsible for the MSC response shown in this study [3, 42, 43]. MSCs differentiated into the chondrogenic lineage may display more surface integrins present, thus facilitating their response to the mechanical environment [44, [45]46]. Alternatively, the ECM produced during culture may have contributed to the better transfer of mechanical stimulation to the cells and to enhanced cell signaling while loading. In this respect, the scaffolding protein p130Cas may have played a key role in cell mechanotransduction, as proposed by Sawada et al. [47] and Geiger et al. [48]. This sort of cell feedback may have been responsible for the observed further specific cartilage gene expression and matrix production.

The response of embryonic stem cells to mechanical stimulation is largely unknown. A recent study by Saha et al. concluded that human ESC differentiation was inhibited by biaxial cyclic strain [49]. Conversely, Huang et al. showed that hESC differentiation into vascular cells could be appropriately modulated by shear stimulation [28]. The hEBd cells used in this study are a different cell population in that they are partially differentiated and have a number of properties of MSCs including cell surface markers and differentiation capabilities [27]. Similarly, Barberi et al. produced a population of cells from hESCs with mesenchymal progenitor characteristics [50]. We previously demonstrated that the hEBd cells undergo chondrogenesis when encapsulated in hydrogels containing adhesion peptides compared with pellet and PEG hydrogel cultures, where minimal differentiation was observed [27]. Without growth factor conditioning (absence of TGF-β), mechanical compression produced a downregulation of chondrogenic genes such as Sox-9, aggrecan, and type II collagen in the embryonic cells. hEBd cells cultured in chondrogenic conditions with TGF-β1, where they are differentiating toward a cartilage phenotype, exhibited a mechanoresponsive behavior similar to MSCs. After 2 weeks, hEBd cells responded to mechanical compression by upregulating chondrogenic genes, such as Sox-9, aggrecan, and type II collagen, over twofold. TGF-β1 administration was chosen to ensure that hEBd cells started to differentiate into the chondrogenic lineage. Afterward, mechanical stimulation was applied to evaluate any synergistic or antagonistic effect with chemical conditioning. From our findings, it appears that chemical cues (TGF-β1) are a prerequisite to appropriately differentiate hEBd cells and benefit from a synergistic effect with mechanical stimulation when this is applied (Figs. 4, 5). It still remains to be determined whether daily mechanical stimulation in conditioned medium would be more effective.

In our system, MSCs responded to mechanical stimulation by upregulation of chondrogenic genes and increased matrix production. The difference in gene response of the hEBd cells with no conditioning (without TGF-β study) and 1-week conditioning versus 2- and 3-week conditioning can be attributed to hEBd cell chondrogenic differentiation. As in the case of MSCs, the positive response to mechanical stimulation after differentiation may be attributed to an increase in cell surface receptors such as mechanosensitive integrins or the presence of ECM molecules to facilitate the physical connections between the mechanical stimuli and the cell [46, 51]. ECM is important in the mechanotransduction process as it is first to interpret forces from the surrounding environment and to transmit these forces to the cell surface (integrins) and then to the cytoskeleton (scaffolding proteins) [52, 53]. The change in the mechanoresponsive behavior of the hEBd cells with TGF-β1 conditioning can be attributed to variations in gene response dependent on the stage of differentiation or changes in force propagation through the ECM and the hydrogel system. Further investigation is needed to clarify the mechanism responsible for the change in mechanoresponsive behavior of hEBd cells and to identify the similarities in mechanical response with MSCs.

In this study, MSCs and hEBd cells were mechanically stimulated with different mechanical protocols. Specifically, a stimulation of 2–2.5 hours appeared to be optimal for both of the cell populations in terms of their chondrogenic differentiation, as shown by the increase in gene expression and/or ECM formation. Although a further increase in MSC aggrecan expression and GAG production was measured for longer stimulation time, this was not supported by a correspondent increase of Sox-9 and, especially, of type II collagen. Whereas Sox-9 is known to be involved and upregulated in early chondrogenesis [54, 55], type II collagen expression might have been hampered by prolonged compression. The increase in the stimulation time, in fact, may have caused a leveling off in cell-cell signaling, which may have limited the production of ECM components to aggrecans only. Moreover, the dynamic stress-strain protocol applied in this study was maintained constant. A change in the applied stress and strain may very well result in a modulation of the ECM produced. Future studies will focus on the optimization of MSC and hEBd cell mechanical stimulation to maximize their chondrogenesis. The ability of embryonic-derived cells to respond to mechanical stimulation in a similar way to MSCs and chondrocytes suggests that not only can they differentiate into chondrocytes, but they are adopting functional properties that are necessary in cartilage tissue: the regulation of a mechanical environment.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

MSCs in hydrogels responded to mechanical stimulation in the absence of TGF-β1 by upregulating chondrogenic genes. When culture medium was supplemented with TGF-β1, MSCs continued to increase chondrogenic gene expression (Sox-9, aggrecan, and type II collagen) as well as ECM production in the presence of mechanical compression.

Cells derived from human embryonic stem cells, the hEBd cells, respond to mechanical stimulation depending on phenotype or extent of chondrogenic differentiation. In the absence of TGF-β1, hEBd cells exhibited a downregulation of chondrogenic gene expression. However, after 2 weeks of TGF-β1 conditioning, mechanical compression increased the chondrogenic differentiation of hEBd cells.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References

This research was funded by the United States-Israel Binational Science Foundation (number 2003184) and the Johns Hopkins University-Technion Program for the Biomedical Sciences and Biomedical Engineering.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References