While the biochemical conditions for stimulating chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs) in vitro have been defined,1 the potential of MSCs to generate neo-cartilage in vivo remains a topic of ongoing research. Animal studies often utilize MSCs from monolayer expansion culture,2–6 a state in which MSCs are undifferentiated. Long-term follow-up has shown that transplanted undifferentiated MSCs in an adult equine model6 and human patients5 have not produced a lasting, hyaline-like tissue. Therefore, it has been suggested that induction of MSC chondrogenesis prior to implantation would promote superior repair.6 In support of this hypothesis, small animal models have shown an increase in cartilage repair when MSC-seeded scaffolds were cultured in chondrogenic medium before implantation in experimental defects.7, 8
Pre-implantation culture of cell-seeded scaffolds is a means by which MSC chondrogenesis may be stimulated. However, ex vivo scaffold seeding is a step that would be otherwise unnecessary for carriers such as hydrogels, which as liquids can be mixed with cell suspensions and then crosslinked within defects9 without the need for additional fixation, and using minimally invasive techniques.6 In order to preserve this feature of injectable scaffolds, pre-implantation differentiation strategies must induce MSC chondrogenesis in systems from which single cells may be obtained. In this study, we address this goal by exploring chondrogenic conditioning of MSCs in suspension cultures from which single cells were readily obtained.
The extent to which chondrogenic commitment of MSCs is necessary to improve cartilage regeneration has yet to be defined, and previous work in vivo has suggested that chondrogenic conditioning may not require extensive ex vivo culture.8 In addition, laboratory studies have reported evidence of MSC chondrogenesis with short periods of differentiation culture.10–12 Given these data, we explored the effect of culturing MSCs in chondrogenic suspension culture for 3 days. MSCs recovered from suspension were immediately transferred to agarose13–15 and evaluated for chondrogenesis relative to undifferentiated MSCs from expansion cultures. In addition, we explored the effect of 2 days of monolayer expansion after suspension conditioning to restore the cell population that was reduced in number during suspension culture. The effects of both protocols were evaluated via extracellular matrix (ECM) synthesis and accumulation, cell viability, and aggrecan and type II collagen gene expression. Furthermore, the single-cell suspensions in agarose allowed for the analysis of the individually encapsulated MSCs.
Bone Marrow Harvest and MSC Expansion
Bone marrow was harvested from the ilium of 15 2- to 5-year-old mixed breed horses, an species and age used for human models of cartilage resurfacing.16 The nucleated cells were isolated via centrifugation and then seeded into low glucose DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) at 0.25 × 106 cells/cm2. The medium was changed after 24 h. MSC colonies proliferated over 6–10 days, and were then reseeded in αMEM (Invitrogen) + 10% FBS + 1 ng/ml fibroblast growth factor 2 (FGF-2) (R&D Systems, Minneapolis, MN) at 12 × 103 cells/cm2. Cultures were cryopreserved after reaching 80% confluence.
Suspension flasks were created by coating 75 cm2 surfaces with 1 ml of 50 mg/ml polyHEME (Sigma-Aldrich, St. Louis, MO), a material that inhibits cell adhesion. Cryopreserved MSCs were thawed and expanded through one passage. 1.8 × 106 MSCs were seeded into suspension flasks in defined medium (high glucose DMEM (Invitrogen, Carlsbad, CA) plus 1% ITS+ (Sigma-Aldrich), 0.1 µM dexamethasone (Sigma-Aldrich), and 37.5 µg/ml ascorbate-2-phosphate (Wako Chemicals, Richmond, VA) plus 10 ng/ml recombinant human transforming growth factor beta 3 (TGFβ) (R&D Systems, Minneapolis, MN).1 After 3 days, MSCs in suspension cultures were collected, rinsed, and exposed to 0.25% trypsin/EDTA to obtain a single cell suspension.
MSC Seeding in Agarose
MSCs were resuspended in warm 2% (w/v) low melting temperature agarose (Invitrogen) at 10 × 106 cells/ml. The cell suspension was pipetted onto the surface of a Petri dish, and cooled to induce gelation.
Live and dead cells were identified using a calcein/ethidium bromide cell viability kit (Invitrogen). Biopsies were harvested from the MSC-seeded agarose and analyzed via visual inspection using a fluorescent microscope. Quantification was performed by counting at least 100 live and dead cells across numerous fields.
For the final 24 h of each agarose culture, 10–20 mg samples were labeled in medium supplemented with 5 µCi/ml 35S-sulfate and 10 µCi/ml 3H-proline, and then digested in proteinase K solution to measure the rate of synthesis of sulfated proteoglycans and total protein, respectively. From the digests, the total sulfated glycosaminoglycan (GAG) content was measured via the DMMB dye binding assay. All data were normalized to wet weight.
Agarose samples were incubated in calcein, and then 0.0005% toluidine blue solution in PBS for 30 min. Samples were microscopically imaged for viable cells with fluorescence, while proteoglycan staining was imaged using light. Quantification of the percentage of viable cells that had secreted an abundant, proteoglycan-rich ECM that stained with toluidine blue was performed by counting at least 100 cells across numerous fields.
Samples were frozen in OCT, sectioned (5 µm thick), and mounted onto slides. Sections were incubated in chondroitinase ABC (Sigma–Aldrich) for 15 min, washed, and blocked with normal goat serum solutions (Jackson Immunoresearch, Westgrove, PA) for 1 h at room temperature. Sections were then incubated in type II collagen (Developmental Studies Hybridoma Bank, University of Iowa) primary antibody supernatant for 1 h, and then rinsed three times in PBS. The sections were incubated in a FITC-conjugated secondary antibody solution (Jackson Immunoresearch) for 1 h, rinsed three times, and then mounted with a coverslip. Slides that were not exposed to the primary antibody were stained with secondary antibody as a control.
RNA Extraction and PCR
RNA was extracted from monolayer and suspension cultures with RLT buffer (Qiagen, Chatsworth, CA) and purified using the RNeasy Mini Kit (Qiagen). MSC-seeded agarose samples were pulverized, stabilized in Trizol (Invitrogen), homogenized, and then centrifuged at 10,000g. The supernatant was collected, and 20% chloroform (v/v) was added. Following centrifugation for 15 min at 12,000g, the supernatant was collected, and RNA was purified using the RNeasy Mini Kit (Qiagen). Two hundred sixty out of 280 nm absorbance measurements were read on a spectrophotometer (NanoDrop Technologies, Wilmington, DE) to determine concentration and purity. RNA was reverse-transcribed to cDNA in 50 µM random hexamers (Superscript III cDNA synthesis kit, Invitrogen). Semi-quantitative real-time PCR was performed using the Applied Biosystems 7000 system and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). Primers and probes for type I and II collagen, aggrecan, and 18S were purchased from the Lucy Wittier Molecular & Diagnostic Core Facility, University of California, Davis. For each sample, expression was normalized to 18S threshold values. Data for each condition was organized by gene, and was calibrated to average expression in monolayer cultures.17
A timeline of experiments is presented in Figure 1. Experiments evaluating chondrogenesis following suspension culture are denoted as “suspension,” with experimental samples abbreviated as “Susp.” Experiments that included 2 days of monolayer expansion in αMEM plus FGF-2 following suspension culture are denoted as “suspension/expansion,” with experimental samples abbreviated as “Susp/Exp.” Experimental and undifferentiated preparations were seeded in agarose and cultured in the presence (TGFβ+) or absence (TGFβ−) of 10 ng/ml TGFβ to test for differentiation in conditions that support strong or minimal chondrogenesis, respectively. Cell viability in agarose was recorded at 0 and 15 days, while all measures of ECM synthesis were evaluated at the end of the experiment. The suspension/expansion protocol was repeated in order to explore gene expression at the different stages of preparation and differentiation. Samples were collected from undifferentiated expansion culture prior to seeding in suspension, after 3 days of suspension culture, after 2 days of expansion culture, and following 15 days of culture in agarose.
Each protocol was evaluated by conducting independent experiments using MSCs from three or five animals, with different donors used for each experiment. ECM synthesis, viability, and gene expression data were calculated from mean values for each donor animal, resulting in a sample size of three or five. Data were analyzed using a mixed model analysis of variance (SAS, SAS Institute, Inc., Carey, NC), with the donor animal used as a random effect. Individual comparisons were made using least square means procedure. p-Values <0.05 were considered significant. Data were reported as mean ± SEM.
After 12 h, MSCs had aggregated into spheres ranging between 25 and 200 µm diameter (data not shown). Cell debris in the medium suggested that not all MSCs survived the early stages of suspension culture. After 3 days, viability staining showed some cell death within the aggregates (data not shown). From 2.4 × 106 cells seeded into suspension for 10 donor animals, 1.14 ± 0.06 × 106 MSCs were recovered.
ECM synthesis: In controls, 3H-proline and 35S-sulfate incorporation and GAG accumulation in TGFβ+ medium were 12-, 33-, and 16-fold higher than TGFβ−, respectively (p < 0.01, Fig. 2). ECM synthesis in Susp-TGFβ− cultures was similar to Control-TGFβ− (p = 0.12–0.82). In TGFβ+, 3H-proline incorporation and GAG accumulation in suspension cultures were 78% and 67% of controls, respectively, although these difference were not significant (p = 0.31, 0.14). 35S-sulfate incorporation in suspension samples was 44% of controls (p < 0.01). MSC viability: MSCs in agarose were >95% viable on day 0. On day 15, viability was significantly different among conditions (p < 0.05). Susp-TGFβ+ retained the highest viability (84%). Viability in Control-TGFβ+ (55%) was greater than Susp-TGFβ− (48%), while the lowest viability was in Control-TGFβ− samples (31%).
Proliferation following suspension culture: From the 2.4 × 106 cells seeded for suspension cultures established with MSCs from for five donor horses, 0.93 ± 0.07 × 106 MSCs were recovered. Over 2 days of monolayer culture, the recovered MSCs proliferated to 3.23 ± 0.45 × 106 MSCs. ECM synthesis: In controls, 3H-proline and 35S-sulfate incorporation and GAG accumulation in TGFβ+ were 31-, 52-, and 10-fold higher than TGFβ−, respectively (p < 0.005, Fig. 3). ECM synthesis in Susp/Exp-TGFβ− cultures was higher than Control-TGFβ− (3H-proline: 5.5-fold, 35S-sulfate: 9.6-fold, GAG accumulation: 3.7-fold; p < 0.005). However, ECM synthesis in Susp/Exp-TGFβ− was 18–37% of Control-TGFβ+ (p < 0.005). ECM synthesis in Susp/Exp-TGFβ+ and Control-TGFβ+ were not significantly different (p = 0.67–0.82). MSC viability: MSCs in agarose were >95% viable on day 0. On day 15, viability was highest in Susp/Exp-TGFβ+ (76%, p < 0.01). Susp/Exp-TGFβ− (59%) and Control-TGFβ+ (55%) were not significantly different (p = 0.08), while the lowest viability was in Control-TGFβ− (31%, p < 0.05). ECM Staining: Toluidine Blue: Sequential incubation in calcein and toluidine blue was sufficient to stain both viable cells and proteoglycan matrix (Fig. 4). In Control-TGFβ− samples, toluidine blue staining was nearly absent, with the occasional finding of a viable MSC surrounded by toluidine blue-positive ECM (Fig. 4A,B). In Susp/Exp-TGFβ−, toluidine blue-positive MSCs were more numerous than in Control-TGFβ− cultures (Fig. 4C), although many viable cells had not accumulated a proteoglycan-rich ECM (Fig. 4D). In Control-TGFβ+ and Susp-TGFβ+ cultures, proteoglycan staining was more frequently observed than in TGFβ− cultures (Fig. 4E,G), although both conditions also contained a population of viable cells that were surrounded by little or no ECM (Fig. 4F,H). The percentage of viable cells surrounded by an abundant proteoglycan-rich ECM were quantified for samples from three donor horses (Fig. 4I), with significant differences among all conditions (p < 0.05). The highest frequency of abundant toluidine blue staining was found for Control-TGFβ+ samples (60%), followed by Susp/Exp-TGFβ+ (40%), Susp/Exp-TGFβ− (14%), and Control-TGFβ− (1.6%). Type II collagen immunohistochemistry: Susp-TGFβ− samples were evaluated for type II collagen accumulation, with samples that accumulated a relatively high (1.53 µg/mg) and low (0.46 µg/mg) amount of GAG presented in Figure 5. Type II collagen was found in pericellular regions of ECM accumulation, as was observed for toluidine blue staining, with a greater frequency of type II-positive MSCs in the high GAG section (Fig. 5A) relative to the low GAG sample (Fig. 5B). No staining was detected in control sections (insets). Gene expression: The lowest expression of ECM was found for undifferentiated MSCs (Fig. 6). Aggrecan: Following suspension culture, aggrecan expression increased 460-fold (p < 0.05). After subsequent monolayer culture, aggrecan expression decreased 8.2-fold (p < 0.05), although expression remained 56-fold higher than undifferentiated MSCs (p < 0.05). In agarose control cultures, aggrecan expression was at least 3,500-fold higher than undifferentiated monolayer. In suspension/expansion agarose samples, aggrecan expression was ∼40- and 330-fold higher than that following suspension and postsuspension expansion, respectively (p < 0.05). Among agarose samples, aggrecan expression was similar (p = 0.09–0.82) except for Susp/Exp-TGFβ−, which was ∼7-fold higher than both control cultures (p < 0.05). Type II collagen: Type II collagen expression increased 181-fold with suspension culture, and then decreased to threefold higher than undifferentiated culture after postsuspension expansion (p < 0.05). In control agarose cultures, type II collagen expression was at least 315-fold higher than undifferentiated MSCs (p < 0.05), with similar expression levels between TGFβ conditions (p = 0.37). Type II collagen expression in suspension/expansion agarose samples were >3.6- and 194-fold higher than Susp and Susp/Exp, respectively, with similar expression levels between TGFβ conditions (p = 0.61). Type I collagen: Type I collagen expression increased ninefold with suspension culture (p < 0.5), and then decreased to values that were similar to undifferentiated cultures following postsuspension expansion (p = 0.20). In agarose samples, type I collagen expression was similar among all conditions (p = 0.37–0.75), at ∼5-fold higher than undifferentiated MSCs p < 0.05).
We explored MSC chondrogenesis following short-term exposure to chondrogenic medium in suspension culture, a technique that resulted in cell aggregates that resembled pellet cultures that are used to study chondrogenesis.1, 10, 18 Importantly, the aggregates readily dissociated in trypsin, allowing for the evaluation of chondrogenesis of the single cell suspension. Suspension-conditioned MSCs cultured in agarose without TGFβ synthesized ECM at a rate that was similar to controls despite a modest increase in cell viability, demonstrating that suspension conditioning alone did not stimulate chondrogenesis. Furthermore, 35S-sulfate incorporation in TGFβ+ samples suggested that suspension conditioning reduced the chondrogenic potential of MSCs, a finding that resembled 35S-sulfate incorporation of MSCs recovered from and reseeded into alginate.19 These data were unlike reports of increased chondrogenesis in studies where TGFβ was withdrawn from chondrogenic cultures,10–12 and suggest that suspension conditioning followed by aggregate dissociation is not a promising technique for enhancing MSC-based cartilage repair.
Suspension cultures led to significant cell death, a result that was consistent with a previous study that demonstrated a decrease in viability of adult equine MSCs in chondrogenic agarose cultures.20 Furthermore, additional examples of MSC necrosis and/or apoptosis in chondrogenic medium have been previously reported.21–24 Given the loss of cells with suspension culture, we restored the cell population with 2 days of monolayer expansion. Suspension/expansion conditioning proved capable of inducing chondrogenesis in the absence of TGFβ as ECM synthesis in Susp/Exp-TGFβ− samples was higher than Control-TGFβ− cultures, and type II collagen accumulation was identified in Susp/Exp-TGFβ− cultures. In TGFβ+ cultures, similar ECM synthesis between control and suspension/expansion samples demonstrated that suspension conditioning did not adversely affect MSC differentiation in a strongly chondrogenic environment. In addition, similar gene expression of type I collagen among agarose cultures suggested that suspension/expansion did not promote a more fibroblast-like phenotype than conventional methods of inducing MSC chondrogenesis.
Given that MSCs from monolayer expansion possess an undifferentiated phenotype,18 it is noteworthy that expansion following suspension culture was necessary to induce cartilage-like ECM synthesis without additional exposure to TGFβ. Based on this finding, we explored gene expression markers of the chondrocyte phenotype during suspension/expansion culture to better understand the influence of each conditioning step. Suspension culture significantly increased the expression of aggrecan and type II collagen over undifferentiated cultures, although the decrease in aggrecan and type II collagen expression with subsequent expansion indicated de-differentiation toward, but not to, the undifferentiated phenotype. Based on these data, stimulation of ECM synthesis with postsuspension expansion could not be attributed to an enhancement of chondrocyte-like differentiation during monolayer culture.
A second factor that may be associated with the difference in chondrogenesis between suspension and suspension/expansion conditioning may be disruption of the cell aggregates following suspension culture. MSC chondrogenesis is associated with temporal changes in gene expression and protein synthesis with differentiation, with numerous differences in ECM gene and protein expression over the first 6 days of culture.18 In addition, chondrogenesis of mesenchymal cells has been found to be sensitive to N-cadherein expression25 and Notch signaling,26 with transient activity during the initial days of chondrogenic culture required for subsequent synthesis of cartilage-like ECM. The three-dimensional environment of a pellet culture or scaffold is an important factor in promoting MSC chondrogenesis, and it is possible that dissociation of the cell aggregates disrupted critical processes that were not sustained upon immediate transfer to agarose. Therefore, despite the induction of chondrocyte-like gene expression in suspension culture, subsequent chondrogenesis in agarose may have been compromised by dissociation of the cell aggregates. Conversely, the partial de-differentiation with postsuspension expansion may have allowed for the recapitulation of those chondrogenic signals associated with physical environment. Additional studies are needed to better characterize the sensitivity of MSCs to the initiation of chondrogenesis as the events that take place early in differentiation may restrict the amount of permissible manipulation.
Quantification of the frequency of MSC differentiation to a phenotype that secreted an abundant cartilage-like ECM revealed a heterogeneous propensity for differentiation of the viable cell population, a detail that has not been previously reported. The potential for variability of chondrogenic differentiation within MSC preparations is supported by numerous studies that have identified markers of heterogeneity within culture-expanded MSC populations.27–29 Here, control samples cultured without TGFβ demonstrated a very low incidence of spontaneous differentiation to a highly active phenotype, as previously observed in photopolymerizing hydrogel cultures,30 although many cells remained viable. In addition, continuous exposure of control samples to TGFβ was not capable of inducing abundant ECM synthesis in all viable cells. Similarly, suspension/expansion cultures revealed heterogeneity in the propensity of viable MSCs to differentiate into a highly active phenotype. While quantification of MSCs that secreted an abundant ECM does not necessarily represent the entire population of accumulated proteoglycans, the large halos of toluidine blue staining around certain MSCs suggest that measures of proteoglycan synthesis were strongly influenced by these highly active cells. Therefore, in Susp/Exp-TGFβ− samples, it is likely that the increase in ECM synthesis over Control/TGFβ− was attributed to a greater presence of MSCs that spontaneously differentiated into a phenotype that secreted an abundant ECM. In TGFβ+ cultures, suspension/expansion culture contained a greater frequency of MSCs that secreted little to no ECM than did control samples, a result that likely explains why the 40% increase in cell viability with suspension/expansion conditioning did not result in higher ECM synthesis.
In agarose cultures, the 3,500- and 300-fold increase in gene expression of aggrecan and type II collagen, respectively, in Control-TGFβ− samples relative to undifferentiated monolayer culture demonstrated that defined medium plus dexamethasone was sufficient to induce chondrocyte-like expression, as has been reported for human MSCs in pellet culture31 and equine MSC-seeded agarose15 after 21 days of culture. Aggrecan and type II collagen expression in all agarose cultures were largely similar, a result that was in contrast to the higher rates of ECM synthesis in TGFβ+ cultures. TGFβ is known to play an important role during early events in progenitor cell condensation and chondrogenesis,32 and exposure of MSCs to TGFβ in an undifferentiated state may be necessary to stimulate lasting ECM synthesis, as reported for chick limb mesenchymal cells.33 Aggrecan and type II collagen gene expression following postsuspension expansion demonstrated a low level of chondrocyte-like differentiation. Therefore, while the analysis of differences among subpopulations within each MSC preparation was beyond the scope of this study, it is possible that a subset of MSCs retained a state of differentiation that inhibited the transition to a highly active phenotype with TGFβ.
In this study, we explored the influence of suspension conditioning in weak (TGFβ−) or strong (TGFβ+) chondrogenic environment. Such in vitro studies provide important information on the potential of MSC preparations to undergo chondrogenesis in a controlled environment, although testing in vivo is necessary to understand the effect of a pre-implantation, chondrogenic conditioning within the joint. Based on animal studies conducted with undifferentiated MSCs that showed early signs of cartilage-like repair tissue, the joint appears to provide at least a limited chondrogenic environment. Therefore, while suspension/expansion conditioning induced high levels of ECM synthesis in only a subset of cells in TGFβ− culture, it is possible that the joint environment may provide additional chondrogenic cues that enhance neo-tissue deposition beyond that which was observed here. However, should animal studies prove that more extensive chondrogenic differentiation is necessary to heal cartilage, it is possible that improvements in overall chondrogenesis may be obtained through a better understanding of factors associated with manipulation of MSCs during early-stages of chondrogenesis as well as the heterogeneous propensity for differentiation to a highly active phenotype.
Funded through a grant from the CSU College Research Council.