TY - JOUR
AU - Barabino, Gilda A.
AB - Abstract Controlled differentiation of mesenchymal stem cells (MSCs) into the chondrogenic lineage is crucial for in vitro generation of neocartilage, yet achieving it remains challenging. Traditional protocols for MSC differentiation using exogenous inductive molecules, such as transforming growth factor-β, fall short in meeting the needs of clinical applications because they yield differentiated cells that exhibit hypertrophic characteristics and subsequently facilitate endochondral bone formation. The objective of the current study was to deliver endogenous inductive factors from juvenile articular chondrocytes to bone marrow-derived MSCs to drive MSC chondrogenic differentiation through cocultivation of the two cell types in the absence of direct physical contact and exogenous stimulators. An initial chondrocyte/MSC ratio of 63:1 was identified as the appropriate proportion of the two cell populations to ensure that coculture-driven MSC-differentiated (CDMD) cells replicated the cellular morphology, behavior, and phenotype of articular chondrocytes. In a three-dimensional agarose system, CDMD cells were further shown to develop into robust neocartilage structurally and mechanically stronger than chondrocyte-laden constructs and with reduced hypertrophic potential. Although MSCs tended to lose the ability to express CD44, an important regulator in cartilage biology, during the coculture induction, CDMD cells regained this function in the three-dimensional tissue cultivation. The present work establishes a chondrocyte/MSC coculture model that serves as a template to better understand chondrocyte-driven MSC differentiation and provides insights for improved strategies to develop clinically relevant cartilage tissue replacements. Tissue regeneration, Mesenchymal stem cells, Chondrogenesis, Serum-free, Hypertrophy Introduction Mesenchymal stem cells (MSCs) derived from different origins, such as bone marrow [1], adipose tissues [1], synovial membranes [1, 2], or umbilical cord blood [3], are attractive for repair and regeneration of tissue or organ defects because of their multipotency and expandable life span. Transplantation of MSCs into rabbit [4] or porcine [5] defective knee joints to restore cartilage function has met with some success, yet the use of MSCs for cartilage repair is still at the preclinical and phase I stages [6]. Wider use of MSCs is limited by the lack of ex vivo techniques to precisely control MSC differentiation into a desired cell lineage. To overcome this hurdle, a thorough understanding of in vivo MSC differentiation mechanisms is required [7, 8]. In vitro, several bioactive agents, such as transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2), and basic fibroblast growth factor (FGF-2), have been used to direct MSC chondrogenic differentiation [9] with TGF-β (TGF-β1, -β3) shown to be essential and widely used [10–15]. When cells are encapsulated in three-dimensional (3D) substrates, however, TGF-β-treated MSC cultures exhibit functional properties inferior to those of chondrocyte-laden constructs [13, 15]. Exposure of MSCs to multiple rather than a single inductive molecule may lead to a higher level of chondrogenesis [16]. Recently, a medium cocktail consisting of TGF-β1, IGF-1, and FGF-2 was shown to promote more robust chondrogenesis of synovium-derived MSCs compared with TGF-β1 alone [11, 14]. This evidence confirms that a single inductive molecule may have limited capacity to trigger specific differentiation pathways because of the complex crosstalk between chemical signals during differentiation, and that the synergism between these signals is required to finalize cell fate [7, 8]. The most straightforward and common way to deliver soluble factors to culture systems is through direct addition of bulk molecules to culture media [11, 13–15]. Macdonald et al. suggest that the bulk and quick release of inductive molecules may compromise cell differentiation and that gradual delivery of those biomolecules to target cells can more closely replicate in vivo conditions [17]. Among the approaches for gradual delivery of inductive agents to cells are gene therapy [12] and polymeric vehicles for molecule release [17, 18]. Although they are promising, these methods are currently limited by economic and technical constraints. Primary chondrocytes can serve as an effective and economic inducer of MSC chondrogenesis, given their ability to gradually secrete a variety of protein molecules, including TGF-β [19, 20], IGF-1 [21, 22], BMP-2 [23, 24], and FGF-2 [25], which may account for in vivo MSC-assisted cartilage regeneration in the absence of exogenous inducers [5]. Traditional protocols with exogenous induction of in vitro MSC chondrogenic differentiation produce cells that typify hypertrophic chondrocytes, which express a relatively significant level of type X collagen, instead of type II [8, 26], and alkaline phosphatase (ALP), which facilitates endochondral bone formation [5, 26, 27]. If MSCs are directly transplanted in vivo without going through a traditional in vitro induction process, one would expect that ossification of implants and adjacent tissues would be eliminated [5]. It may be that the surrounding chondrocytes also play a role in stabilizing the phenotype of MSC implants via paracrine regulation. The primary objective of the current study was to promote neocartilage development through cocultivation of bone marrow-derived MSCs with juvenile articular chondrocytes serving as a feeder of morphogenetic signals, to induce chondrogenic differentiation of MSCs. In the absence of biomolecular factors such as serum and exogenous growth factors and without direct physical contact, we found that at a chondrocyte/MSC ratio of 63:1, our in vitro coculture model closely resembled in vivo conditions and the derived MSC-differentiated cell population mimicked articular chondrocytes. In addition, coculture-driven MSC-differentiated (CDMD) cells grown in a 3D hydrogel system developed into robust neocartilage that resisted hypertrophic maturation and calcification. Materials and Methods Materials Unless specified otherwise, supplies and reagents were purchased from VWR International (West Chester, PA, http://www.vwr.com), Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com), or Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Antibodies were from AbD Serotec (Raleigh, NC, http://www.ab-direct.com) or Abcam (Cambridge, MA, http://www.abcam.com). Cell Isolation Bovine chondrocytes and bone marrow MSCs were isolated from freshly slaughtered 2-week-old calves. Briefly, articular cartilage sections were harvested from the femoropatellar groove, followed by digestion with type II collagenase in Dulbecco's modified Eagle's medium (high-glucose DMEM). The supernatant of the digested tissues was centrifuged to obtain chondrocyte pellets. Isolated chondrocytes were stored in liquid nitrogen for later use. Bone marrow MSCs were harvested from the portion of red bone marrow within the trabecular bone in femurs of donors. Briefly, isolated marrow was mixed with the expansion medium (EM; DMEM supplemented with 10% certificated fetal bovine serum [FBS] and 1× penicillin-streptomycin-fungizone [PSF]) supplemented with 300 U/ml heparin. After vortexing to remove fat from the marrow, the supernatant was passed through a 70-μm cell strainer. The resulting solution was centrifuged to collect cell pellets. Cells were resuspended in the EM and plated onto T-225 flasks. After an initial period of 72 hours, nonadherent cells were removed from the flasks, whereas adherent cells were cultured with the EM for an additional 7–10 days until cultures reached confluence. Subsequent subculturing was carried out, with cultures up to passage 4 used in the present study. Passaged MSCs were frozen in liquid nitrogen. Coculture of MSCs with Chondrocytes Prior to each experiment, cells isolated from at least three donors were pooled together. A small aliquot of passage 4 MSCs was subjected to flow cytometry analyses to determine the purity of the population. Supplemental online Figure 1 shows that the majority of passage 4 cells were positive for CD166, CD44, and CD271, three of the major mesenchymal markers, but negative for CD45, a hematopoietic marker [28]. To set up a noncontact coculture system, 5 × 104 live MSCs were seeded on the surface of each of tissue culture wells and allowed to settle for 24 hours. Conversely, primary chondrocytes with varied numbers were centrifuged in Eppendorf tubes to form cell pellets, which were later transferred to Transwell inserts (Corning, Corning, NY, http://www.corning.com) after an initial period of 24 hours. The two compartments were assembled as shown in Figure 1A and thereby shared the same culture medium through a 0.4-μm transmembrane that prevented cell migration but allowed small molecule exchange. Coculture was then initiated. Five different initial ratios of the two cell populations were examined, and two control groups consisting of either chondrocyte pellets or monolayer MSCs were used (Fig. 1B). The coculture groups were divided into three categories: high, medium, and low chondrocyte contents. In a pilot study, four chondrocyte/MSC ratios of less than 7:1 were studied, yet no induction was detected in monolayer MSCs. Cells and pellets were cultivated with a serum-free, growth factor-free inductive medium (IM: DMEM supplemented with 3.6 mg/ml sodium bicarbonate, 10 mM HEPES, 0.4 mM proline, 1% insulin-transferrin-selenium, 1× PSF, 50 μg/ml ascorbic acid, and 0.1 μM dexamethasone) inside a humidified incubator (37°C, 5% CO2) for 15 days. The IM was completely renewed every 3 days. At the end of the cultivation, monolayer cells were harvested for evaluation. Figure 1 Open in new tabDownload slide Cocultivation of bone marrow-derived MSCs with juvenile chondrocytes. (A): Schematic diagram of the chondrocyte/MSC coculture setup. (B): Experimental groups in coculture. aInitial ratios were based on cell numbers. bPrimary chondrocytes were cultured in pellets formed by centrifugation. cPassage 4 MSCs with a constant cell number (5 × 104 cells per well) were cultivated in a monolayer separated from chondrocyte pellets. dCoculture groups were divided into three categories based on chondrocyte contents in culture. (C): LIVE/DEAD (top row) and corresponding phase contrast (bottom row) images of monolayer cells after 15 days in cocultivation (magnification, ×10). Live and dead cells were stained with green and red fluorescent dyes, respectively. Scale bar = 50 μm. Abbreviation: MSC, mesenchymal stem cell. Figure 1 Open in new tabDownload slide Cocultivation of bone marrow-derived MSCs with juvenile chondrocytes. (A): Schematic diagram of the chondrocyte/MSC coculture setup. (B): Experimental groups in coculture. aInitial ratios were based on cell numbers. bPrimary chondrocytes were cultured in pellets formed by centrifugation. cPassage 4 MSCs with a constant cell number (5 × 104 cells per well) were cultivated in a monolayer separated from chondrocyte pellets. dCoculture groups were divided into three categories based on chondrocyte contents in culture. (C): LIVE/DEAD (top row) and corresponding phase contrast (bottom row) images of monolayer cells after 15 days in cocultivation (magnification, ×10). Live and dead cells were stained with green and red fluorescent dyes, respectively. Scale bar = 50 μm. Abbreviation: MSC, mesenchymal stem cell. Live/Dead Fluorescence Prior to the harvest, cells in monolayer were evaluated with a LIVE/DEAD viability dye mixture that was freshly prepared in phosphate-buffered saline (PBS) with a final concentration of 4 mM Calcein AM and 2 mM ethidium homodimer-1. Before staining, cells were washed with PBS to remove medium esterase activity that may increase extracellular fluorescence by hydrolyzing Calcein AM. Monolayer cells were then fixed and incubated with the dye mixture. The fluorescent images were captured under a fluorescence microscope. Quantitative Real-Time Polymerase Chain Reaction Real-time polymerase chain reaction (qRT-PCR) was used to quantify gene expression in harvested monolayer cells. Cells were fixed in TRIzol, and RNA was isolated from the homogenized cell lysate through a series of rinse, elution, and centrifugation. The RNA samples were then reverse transcribed into cDNA using a QuantiTech Rev Transcription kit (Qiagen, Hilden, Germany, http://www.qiagen.com) according to the manufacturer's protocol. Gene expression of interest was quantified on the basis of the SYBR Green real-time telomeric repeat amplification protocol. Custom primers [29, 30] (supplemental online Table 1) for bovine samples were fabricated (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The fluorescent signals were amplified and detected using an ABI Step One Plus sequence detector (Applied Biosystems). The reaction consisted of an initial enzyme activation for 10 minutes at 95°C, followed by 40 or 60 cycles of 15 seconds at 95°C and 1 minute at 60°C. The cycle threshold (Ct) value for each sample was averaged from triplicates. A 2−ΔΔCt approach [31] was used where the fluorescent signals were normalized to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and the MSC control group was used as the reference group. Flow Cytometry Changes in surface antigens of cultured monolayer cells were investigated using flow cytometry. Briefly, a minimum of 1 million cells derived from each group was fixed, treated with a nonspecific blocking agent for 30 minutes, and incubated with fluorescein isothiocyanate-conjugated mouse anti-human CD166 or phycoerythrin-conjugated mouse anti-bovine CD44 antibodies for 1 hour. The anti-human CD166 antibody possesses an interspecies reactivity and is applicable to bovine samples [28]. Antibodies against mouse IgG were used as the negative isotype controls. Flow cytometry was performed in a FACScan (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). The size and granularity of cells were also evaluated using the forward scatter and side scatter parameters, respectively. Long-Term Tissue Cultivation In order to evaluate cell functionality, CDMD cells were encapsulated in agarose hydrogels to grow tissue-engineered cartilage. CDMD cells are defined as the monolayer cells derived from the coculture model with an optimized chondrocyte/MSC ratio (63:1) determined in the cocultivation experiment. To fabricate tissue constructs, primary chondrocytes or CDMD cells were well mixed with an equal volume of 5% (wt/vol) agarose (type VII) solution at 40°C, yielding a seeding density of 40 million live cells per milliliter of 2.5% agarose solution. The mixture was allowed to gelatinize at room temperature, and circular constructs (5 mm in diameter × 2 mm thick) were cored. The cell-laden discs were allowed to stabilize in media for 24 hours and then cultivated with a regular chondrocyte medium (CM: DMEM supplemented with 3.6 mg/ml sodium bicarbonate, 10 mM HEPES, 0.4 mM proline, 0.1 mM nonessential amino acids, 10% FBS, 1× PSF, and 50 μg/ml ascorbic acid) [32] in tissue culture plates for 4 weeks. The CM was changed every 3 days, and constructs were harvested at specific time points. Biomechanics Equilibrium compressive moduli of engineered constructs were determined using an unconfined compression test [33]. During the examination, a preload of 0.01 N was applied to samples until equilibrium was achieved. A stress relaxation test was then carried out at strains of 5%, 10%, 15%, 20%, and 25%, after which samples were allowed to equilibrate. The equilibrium moduli were obtained from the slope of the plot of equilibrium force normalized to the cross-sectional area of the construct versus the strains. Biochemistry Prior to biochemical assays, constructs were lyophilized using a speed vacuum (Labconco, Kansas City, MO, http://www.labconco.com) for 24 hours, weighed (dry weight), and digested with papain enzyme (Worthington Biochemical, Lakewood, NJ, http://www.worthington-biochem.com). DNA was quantified using a PicoGreen dsDNA kit, and the manufacturer's instructions were followed. Sulfated glycosaminoglycan (GAG) contents were assessed spectrophotometrically at 525 nm using a 1,9-dimethylmethylene blue dye binding assay [34]. Chondroitin sulfate was used to create a standard curve and a ratio of chondroitin sulfate to GAG of 1 was assumed. Total collagen contents were determined using an orthohydroxyproline (OHP) colorimetric assay, assuming a 1:10 ratio of OHP to collagen concentration [35]. The GAG and collagen contents are presented in values normalized to the dry weight or the DNA weight of constructs. Alkaline Phosphatase Assay ALP activity was determined and used as an index of hypertrophic potential of engineered constructs. Briefly, one half of each of the samples was lysed and homogenized in Triton X-100 lysis buffer, followed by the 15-minute centrifugation at 10,000g at 4°C. The supernatant was collected for detection of ALP activity. The other half was assayed to obtain the corresponding DNA content. A p-nitrophenyl phosphate (pNPP) approach was used to quantify ALP, where tissue lysate and standards (AnaSpec, Fremont, CA, https://www.anaspec.com) were incubated with pNPP substrate for 45 minutes at room temperature and the reaction was terminated with 0.1 M NaOH. The absorbance was read at 405 nm, and the ALP amounts were normalized to the corresponding DNA contents. Enzyme-Linked Immunosorbent Assay of CD44 CD44 expressed by chondrocytes or CDMD cells within constructs was quantified using an enzyme-linked immunosorbent assay protocol. Briefly, tissue lysate was prepared as described in the ALP assay and diluted in the diluent buffer. The diluted samples were added to each well of 96-well microplates coated with capture anti-CD44 antibodies and incubated for 2 hours. Each well was then treated with biotinylated detection antibodies for another 2 hours. Diluted streptavidin-conjugated horseradish peroxidase (HRP) solution was added to each well and incubated for 30 minutes. Following the 20-minute incubation with tetramethylbenzidine substrate, the enzyme-substrate reaction was stopped by adding H2SO4 to each well. The absorbance was detected at 450 nm. The data are presented as optical density (O.D.) per construct. Histology and Immunohistochemistry In preparation for histology and immunohistochemistry, harvested constructs were fixed, embedded in paraffin, and sectioned in 5-μm-thick slices. The slices were fixed on glass slides, deparaffinized, and then stained with toluidine blue for GAG. Samples were also assessed for ALP activity and mineral deposition using the Sigma-Aldrich 86C kit [36] and the von Kossa method, respectively. Type I and type II collagen and CD44 molecules were labeled using an immunohistochemical method. Briefly, deparaffinized tissue sections were incubated with citrate buffer heated to 99°C for 25 minutes to retrieve antigens, followed by a 20-minute cooling at room temperature. The slides were then incubated with 0.3% hydrogen peroxide for 30 minutes, blocking serum buffer for 20 minutes, and purified rabbit (for collagen) or mouse (for CD44) anti-bovine antibodies overnight at 4°C. Finally, the samples were treated with biotinylated goat anti-rabbit or anti-mouse IgG antibodies for 30 minutes and streptavidin-HRP complex for another 30 minutes, followed by the incubation with diaminobenzidine chromogen reagent until optimal staining was developed. Color images were captured under a light microscope. Statistical Analyses Statistical data are presented as means ± 1 SD and statistical analyses were performed by one-way or two-way analysis of variance in conjunction with the Bonferroni post test for multiple comparisons with significance at a p value of less than .05. Results MSC Chondrogenic Differentiation in Cocultivation Cell Aggregation At the end of the cocultivation, the majority of monolayer cells in all groups survived regardless of the presence and amount of chondrocytes (Fig. 1C). The existence of dead cells was likely because of imperfect initial cell viabilities (80%–85%). When cultivated with the serum-free, growth factor-free IM, cells in the MSC control and low chondrocyte content (chondrocyte/MSC ratio, 7:1) groups exhibited a flatter, irregular morphology and spread out on the surface of the substrate, whereas cells in the other coculture groups tended to form aggregates of different sizes. Qualitatively, the size of such aggregates was observed to increase with increasing chondrocyte number in culture. Gene Expression Gene expression in harvested monolayer cells was quantified using qRT-PCR. Three chondrogenic markers, type II collagen, aggrecan, and SOX9, were evaluated. The expression of collagen II (Fig. 2A) in the high (chondrocyte/MSC ratios, 100:1 and 63:1) and medium (chondrocyte/MSC ratios, 31:1 and 15:1) chondrocyte content groups increased significantly in comparison with the MSC control group (^, p < .05), whereas the highest value was detected in the group with a chondrocyte/MSC ratio of 100:1 (1,240-fold over the MSC control compared with 567-fold, 305-fold, and 14-fold in the 63:1, 31:1, and 15:1 chondrocyte/MSC groups, respectively), which was also higher than the chondrocyte control value (*, p < .05). A similar trend was observed in the aggrecan mRNA level (Fig. 2B), with the greatest expression in the 100:1 chondrocyte/MSC group (105-fold over the MSC control), yet this highest value was not statistically different from the chondrocyte control value and the 63:1 chondrocyte/MSC value (+, p > .05). For SOX9 (Fig. 2C), when compared with both MSC (^, p < .05) and chondrocyte (*, p < .05) control groups, the expression increased significantly in all of the coculture groups except for the 7:1 chondrocyte/MSC group. The difference in SOX9 values between the two high chondrocyte content groups did not achieve statistical significance (+, p > .05). Figure 2 Open in new tabDownload slide Relative gene expression in monolayer cells after 15 days in coculture. Expression was normalized to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), and the MSC control group was used as the reference. (A–C): Chondrogenic markers: type II collagen (A), aggrecan (B), and SOX9 (C). (D, E): Osteogenic markers: type I collagen (D) and RUNX2 (E). (F, G): Hypertrophic markers: type X collagen (F) and MMP-13 (G). *, Statistical significance in comparison with the chondrocyte control (p < .05); ^, statistical significance in comparison with the MSC control (p < .05); +, nonsignificance between the groups (p > .05); n = 6. Abbreviation: MSC, mesenchymal stem cell. Figure 2 Open in new tabDownload slide Relative gene expression in monolayer cells after 15 days in coculture. Expression was normalized to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), and the MSC control group was used as the reference. (A–C): Chondrogenic markers: type II collagen (A), aggrecan (B), and SOX9 (C). (D, E): Osteogenic markers: type I collagen (D) and RUNX2 (E). (F, G): Hypertrophic markers: type X collagen (F) and MMP-13 (G). *, Statistical significance in comparison with the chondrocyte control (p < .05); ^, statistical significance in comparison with the MSC control (p < .05); +, nonsignificance between the groups (p > .05); n = 6. Abbreviation: MSC, mesenchymal stem cell. To exclude the possibility of osteogenic induction in the cocultivation, two osteogenic markers, type I collagen and RUNX2, were studied. The expression of collagen I (Fig. 2D) in the high and medium chondrocyte content groups was significantly lower than that in the MSC control group (^, p < .05). No statistical difference in the collagen I level was determined between the two high chondrocyte content groups (+, p > .05), and the average value of either group was also similar to the chondrocyte control value. Comparable RUNX2 mRNA levels (Fig. 2E) were detected among all the groups (+, p > .05). The hypertrophic phenotype of harvested cells was assessed by measuring the mRNA expression of two hypertrophic markers, type X collagen and MMP-13. For collagen X (Fig. 2F), the elevated level was only observed in the high chondrocyte content groups when compared with the MSC control group (^, p < .05). Notably, the 100:1 chondrocyte/MSC group (209-fold over the MSC control) yielded a significantly higher collagen X value than the chondrocyte control group (154-fold over the MSC control), whereas the 63:1 chondrocyte/MSC value (48-fold over the MSC control) was relatively lower (*, p < .05). For MMP-13 (Fig. 2G), either of the high chondrocyte content groups had an average value similar to that of the chondrocyte control group but significantly greater than that of the MSC control (^, p < .05). Cells in the MSC control and 7:1 chondrocyte/MSC groups exhibited identical expression in all of the studied genes. Conversely, cells in the coculture group with an initial chondrocyte/MSC ratio of 63:1 had a gene expression profile similar to that of the chondrocyte control group, with a relatively reduced hypertrophic phenotype. Changes in Expression of CD166 and CD44 and Morphological Distribution Harvested cells were labeled for two mesenchymal surface markers, CD166 and CD44, and analyzed using flow cytometry. Figure 3A shows that cells derived from the MSC control group maintained a high potential to express both markers (CD166+: 99.4%; CD44+: 99.0%). However, the percentages of CD166+ (top row) and CD44+ (bottom row) populations decreased from 99.3% to 9.20% and from 98.6% to 3.54%, respectively, when the chondrocyte/MSC ratio increased from 7:1 to 100:1. Figure 3 Open in new tabDownload slide Flow cytometric characterization of harvested monolayer cells. (A): Expression of CD166 and CD44 mesenchymal markers in monolayer cells. Cells were labeled with FITC-conjugated anti-CD166 or phycoerythrin-conjugated anti-CD44 antibodies. Filled histograms represent marker molecules. Open histograms indicate negative isotype controls. Percentages of positively stained cell populations are indicated. (B): Morphological distribution of monolayer cells. Percentages of cell populations in region 1 and region 2 are indicated. Abbreviations: FITC, fluorescein isothiocyanate; FSC, forward scatter; MSC, mesenchymal stem cell; R, region; RPE, R-phycoerythrin; SSC, side scatter. Figure 3 Open in new tabDownload slide Flow cytometric characterization of harvested monolayer cells. (A): Expression of CD166 and CD44 mesenchymal markers in monolayer cells. Cells were labeled with FITC-conjugated anti-CD166 or phycoerythrin-conjugated anti-CD44 antibodies. Filled histograms represent marker molecules. Open histograms indicate negative isotype controls. Percentages of positively stained cell populations are indicated. (B): Morphological distribution of monolayer cells. Percentages of cell populations in region 1 and region 2 are indicated. Abbreviations: FITC, fluorescein isothiocyanate; FSC, forward scatter; MSC, mesenchymal stem cell; R, region; RPE, R-phycoerythrin; SSC, side scatter. Flow cytometry also demonstrated the size-granularity distribution of cells (Fig. 3B). In the size-granularity diagram of the MSC control group, two regions were first gated where region 1 indicates the MSC-main area consisting of 87.5% of the entire population, whereas, relative to region 1, region 2 represents a population with smaller cell sizes and slightly lower granularity. The two gated areas were then applied to the other groups. When the chondrocyte/MSC ratio rose from 7:1 to 100:1, cell distribution began migrating from region 1 to region 2. The percentages of cell populations in region 2 were 5.95%, 19.3%, 36.1%, 72.6%, and 75.4% in the 7:1, 15:1, 31:1, 63:1, and 100:1 chondrocyte/MSC groups, respectively. Formation of Chondrocyte-Like Clusters To further validate the change in cellular morphology detected in the flow cytometry studies, harvested monolayer cells were replated onto tissue culture plates at a density of 4,000 cells per cm2 and cultivated with the EM for an additional 4 days. Cells derived from different groups exhibited significantly divergent morphologies (Fig. 4). Specifically, cells harvested from the MSC control and low chondrocyte content groups had a fibroblast-like spindle shape that is commonly observed during MSC expansion processes [37]. Conversely, cells derived from the high chondrocyte content groups formed chondrocyte-like clusters (Fig. 4, dotted circles), whereas combined morphological features were observed in the cultivation of cells obtained from the medium chondrocyte content groups (solid and dashed circles). Figure 4 Open in new tabDownload slide Morphological features of harvested monolayer cells in post-coculture two-dimensional (2D) cultivation (magnification, ×10). Monolayer cells were harvested and recultivated for 4 days with the expansion medium in a 2D environment. A primary chondrocyte group was included for comparison. Dotted (yellow) circles demonstrate chondrocyte clusters. Solid (red) and dashed (green) circles represent chondrocyte- and MSC-specific morphologies, respectively. Scale bar = 50 μm. Abbreviation: MSC, mesenchymal stem cell. Figure 4 Open in new tabDownload slide Morphological features of harvested monolayer cells in post-coculture two-dimensional (2D) cultivation (magnification, ×10). Monolayer cells were harvested and recultivated for 4 days with the expansion medium in a 2D environment. A primary chondrocyte group was included for comparison. Dotted (yellow) circles demonstrate chondrocyte clusters. Solid (red) and dashed (green) circles represent chondrocyte- and MSC-specific morphologies, respectively. Scale bar = 50 μm. Abbreviation: MSC, mesenchymal stem cell. Neocartilage Formation by CDMD Cells Cocultivation of MSCs with primary chondrocytes at a ratio of 63:1 chondrocyte/MSC yielded an MSC-differentiated cell population that best resembled articular chondrocytes in morphology, phenotype, and behavior. Thus, this coculture group was used to generate CDMD cells, whose ability to develop into neocartilage was further investigated and compared with that of primary chondrocytes. Mechanical and Biochemical Properties of Tissue Constructs The equilibrium moduli (Fig. 5A) of both chondrocyte and CDMD constructs were close to the level of cell-free agarose discs (16.8 ± 1.96 kPa) at day 0, yet the values continuously increased thereafter (^, p < .05). Constructs seeded with CDMD cells (22.0 ± 2.67 kPa) were significantly softer than chondrocyte-laden constructs (32.4 ± 2.44 kPa) at day 7 (**, p < .01), whereas both samples exhibited similar stiffness at day 14. At the end of the cultivation, CDMD constructs (81.8 ± 4.15 kPa) demonstrated stronger compressive strength compared with chondrocyte cultures (66.4 ± 4.44 kPa) (***, p < .001). Figure 5 Open in new tabDownload slide Biomechanical and biochemical properties of tissue-engineered cartilage. (A): Equilibrium compressive modulus. (B): DNA content. (C): GAG content. (D): Total collagen content. (E): GAG synthetic activity. (F): Collagen synthetic activity. ^, Significant difference from cell-free agarose discs (p < .05); *, statistical significance between the groups (p < .05); **, statistical significance between the groups (p < .01); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); n = 6. (G): Histological and immunohistochemical images of tissue constructs (magnification, ×10). Samples were stained for GAG (left panel, purple) and type I (middle panel, brown) and II collagen (right panel, brown), respectively. Scale bars = 100 μm. Inset images demonstrate the staining of the corresponding intact constructs. Abbreviations: CDMD, coculture-driven mesenchymal stem cell-differentiated; GAG, glycosaminoglycan; ND, nondetectable. Figure 5 Open in new tabDownload slide Biomechanical and biochemical properties of tissue-engineered cartilage. (A): Equilibrium compressive modulus. (B): DNA content. (C): GAG content. (D): Total collagen content. (E): GAG synthetic activity. (F): Collagen synthetic activity. ^, Significant difference from cell-free agarose discs (p < .05); *, statistical significance between the groups (p < .05); **, statistical significance between the groups (p < .01); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); n = 6. (G): Histological and immunohistochemical images of tissue constructs (magnification, ×10). Samples were stained for GAG (left panel, purple) and type I (middle panel, brown) and II collagen (right panel, brown), respectively. Scale bars = 100 μm. Inset images demonstrate the staining of the corresponding intact constructs. Abbreviations: CDMD, coculture-driven mesenchymal stem cell-differentiated; GAG, glycosaminoglycan; ND, nondetectable. Comparable DNA contents (Fig. 5B) were measured in chondrocyte (4.78 ± 0.67 μg per construct) and CDMD (5.86 ± 1.96 μg per construct) cultures at day 7, whereas the values of chondrocyte-encapsulated constructs (6.00 ± 1.34 and 15.7 ± 3.29 μg per construct, respectively) were compromised when compared with those of CDMD cell-laden constructs (12.7 ± 0.82 and 29.6 ± 5.58 μg per construct, respectively) at days 14 and 28 (***, p < .001). The proliferative rates (supplemental online Table 2) of chondrocytes and CDMD cells within constructs were calculated by normalizing DNA contents at days 7, 14, and 28 to the corresponding day 0 values. The results demonstrated that although similar rates were observed at day 7, the overall proliferative rate of CDMD cells was higher than that of primary chondrocytes, reaching 7.68-fold and 4.82-fold increases in population, respectively, by day 28. The trend of GAG deposition within tissue constructs (Fig. 5C) resembled the profile of equilibrium moduli. Specifically, chondrocyte constructs (10.3 ± 3.04% of dry weight) had a higher GAG content than constructs seeded with CDMD cells (5.77 ± 0.94% of dry weight) at day 7 (**, p < .01). However, more GAG molecules were encapsulated in the extracellular space by CDMD cells thereafter. At day 28, the GAG contents of chondrocyte and CDMD cultures reached 27.0 ± 3.04% and 36.0 ± 2.06% of dry weight, respectively. Although a higher GAG synthetic potential (Fig. 5E) was observed in chondrocytes in comparison with CDMD cells at both day 7 (*, p < .05) and day 14 (**, p < .01), the synthetic activity of CDMD cells continuously increased over time as opposed to levels remaining relatively constant for chondrocytes at days 14 and 28 (+, p > .05). Histologically, GAG deposition (Fig. 5G, left panel) was highly localized in cells at day 7, whereas GAG molecules began occupying the extracellular space after 2 weeks in culture. At day 28, the CDMD samples were more intensely stained for GAG in comparison with the chondrocyte specimens. In the analysis of collagen accumulation (Fig. 5D), CDMD constructs exhibited enhanced collagen contents at both day 14 (4.40 ± 0.42% vs. 2.56 ± 0.28% of dry weight) and day 28 (7.08 ± 0.75% vs. 5.12 ± 0.54% of dry weight) when compared with chondrocyte cultures (***, p < .001). The collagen synthetic activity (Fig. 5F) of CDMD cells was also significantly stronger than that of chondrocytes throughout the cultivation (*, p < .05; ***, p < .001). Histological specimens indicated that the majority of collagen molecules secreted by both cell types were type II and that more intense collagen II staining was observed in the CDMD samples (Fig. 5G, middle and right panels). ALP Activity in Neocartilage The hypertrophic potential of cells within engineered cartilage was assessed by quantification of ALP activity. Figure 6A shows that total ALP contents of either type of constructs continuously increased over time and that, at any time point, the average ALP value of constructs seeded with CDMD cells was compromised compared with chondrocyte-laden constructs (p < .05). When normalized to the corresponding DNA contents (Fig. 6B), the normalized ALP level in CDMD cells remained constant (approximately 5 ng of ALP per μg of DNA) throughout the 4-week cultivation (#, p > .05) and was significantly lower than that in chondrocytes at any time point (***, p < .001). ALP histochemistry (Fig. 6C, left column) revealed that chondrocytes were more intensely stained for ALP in comparison with CDMD cells. In addition, the von Kossa staining (Fig. 6C, right column) showed that no mineral deposition occurred in either group at the end of the tissue cultivation. Figure 6 Open in new tabDownload slide Quantitative and qualitative analyses of ALP activity within tissue-engineered cartilage. ALP was quantified using a p-nitrophenyl phosphate method and the data are presented as ALP content per construct (A) and per microgram of DNA (B). *, Statistical significance between the groups (p < .05); **, statistical significance between the groups (p < .01); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); #, nonsignificance within the group throughout the 28-day cultivation (p > .05); n = 6. (C): Specimens of 28-day constructs were stained for ALP and calcium using the Sigma-Aldrich 86C kit and the von Kossa method, respectively. In ALP histochemistry (left column; magnification, ×60), ALP molecules, nuclei, and cytoplasm were stained blue, dark red, and light red, respectively. Scale bar = 10 μm. In calcium staining (right column; magnification, ×10), mineral components and cytoplasm were stained black and violet red, respectively. Calcified cartilage was used as a control for positive staining. Inset images demonstrate the staining of the corresponding intact constructs. Scale bar = 100 μm. Abbreviations: ALP, alkaline phosphatase; CDMD, coculture-driven mesenchymal stem cell-differentiated. Figure 6 Open in new tabDownload slide Quantitative and qualitative analyses of ALP activity within tissue-engineered cartilage. ALP was quantified using a p-nitrophenyl phosphate method and the data are presented as ALP content per construct (A) and per microgram of DNA (B). *, Statistical significance between the groups (p < .05); **, statistical significance between the groups (p < .01); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); #, nonsignificance within the group throughout the 28-day cultivation (p > .05); n = 6. (C): Specimens of 28-day constructs were stained for ALP and calcium using the Sigma-Aldrich 86C kit and the von Kossa method, respectively. In ALP histochemistry (left column; magnification, ×60), ALP molecules, nuclei, and cytoplasm were stained blue, dark red, and light red, respectively. Scale bar = 10 μm. In calcium staining (right column; magnification, ×10), mineral components and cytoplasm were stained black and violet red, respectively. Calcified cartilage was used as a control for positive staining. Inset images demonstrate the staining of the corresponding intact constructs. Scale bar = 100 μm. Abbreviations: ALP, alkaline phosphatase; CDMD, coculture-driven mesenchymal stem cell-differentiated. CD44 Expression in Neocartilage Since differentiating MSCs were found to possess a reduced potential to express CD44 molecules, a receptor for hyaluronan that highly relates to cartilage tissue properties [38–40], during the cocultivation induction (Fig. 3A), we further evaluated the secretion of CD44 by chondrocytes and CDMD cells in long-term neocartilage development. Figure 7A demonstrates that, at day 0, CD44 expression in CDMD constructs (O.D. = 0.098) was significantly lower than that in chondrocyte constructs (O.D. = 0.268) (***, p < .001) and was close to the level of cell-free agarose samples (O.D. = 0.071) as well as the blank (dashed line, O.D. = 0.064). The intensity of CD44 signals, however, increased over time in both groups. By day 28, the CDMD group (O.D. = 1.46) yielded stronger CD44 signal intensity than the chondrocyte group (O.D. = 1.02) (***, p < .001). CD44 immunohistochemistry (Fig. 7B) showed that, in the beginning of the 4-week cultivation, CD44 staining was relatively evident in chondrocyte cultures, especially at the edge of cells (inset images), but weak in CDMD cells. Yet both chondrocyte and CDMD specimens were intensely stained for CD44 at the end of the culture. Figure 7 Open in new tabDownload slide Quantitative and qualitative analyses of CD44 expression within tissue-engineered cartilage. (A): CD44 molecules were quantified using enzyme-linked immunosorbent assay, and the data are presented as optical density per construct. The dashed line indicates the blank signal derived from the diluent buffer (O.D. = 0.064). ^, Significant difference from cell-free agarose discs (p < .05); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); n = 6. (B): 0-day and 28-day constructs were stained for CD44 using an immunohistochemical method (magnification, ×40). CD44 molecules were stained brown. Inset pictures represent the enlarged images of a single cell. Scale bar = 50 μm. Abbreviations: CDMD, coculture-driven mesenchymal stem cell-differentiated; O.D., optical density. Figure 7 Open in new tabDownload slide Quantitative and qualitative analyses of CD44 expression within tissue-engineered cartilage. (A): CD44 molecules were quantified using enzyme-linked immunosorbent assay, and the data are presented as optical density per construct. The dashed line indicates the blank signal derived from the diluent buffer (O.D. = 0.064). ^, Significant difference from cell-free agarose discs (p < .05); ***, statistical significance between the groups (p < .001); +, nonsignificance within the group at different time points (p > .05); n = 6. (B): 0-day and 28-day constructs were stained for CD44 using an immunohistochemical method (magnification, ×40). CD44 molecules were stained brown. Inset pictures represent the enlarged images of a single cell. Scale bar = 50 μm. Abbreviations: CDMD, coculture-driven mesenchymal stem cell-differentiated; O.D., optical density. Discussion Cocultivation of MSCs with chondrocytes for the in vitro development of neocartilage has been investigated by us and others [41–46], and although it is promising, unresolved issues that limit future potential remain. In most of these studies, MSCs and chondrocytes were mixed in monolayers [43], cell pellets [41, 44], or different types of 3D substrates [42, 45] such that the two cell populations were in close proximity or had direct physical contact. In these cases, data collected in characterization assays represent the summation of signals derived from both cell types rather than from a pure MSC product, making it difficult to distinguish the origin of detected chondrogenic signals. Although additional separation processes, such as fluorescence-activated or magnetic-affinity cell sorting, can be used to isolate the MSC population, such techniques require intense skill and are costly and time-consuming. Moreover, most of the previous coculture experiments relied on TGF-β to ensure the induction of MSC chondrogenesis [41, 42, 44, 45] and therefore do not address whether or not chondrocyte-generated morphogenetic signals can drive MSC differentiation. Conversely, a recent study demonstrated that, when cultivated separately, passaged osteoarthritic chondrocytes could trigger chondrogenesis of human MSCs, p7043L cell line, encapsulated in poly(ethylene-glycol) diacrylate gels, even without exogenous inductive molecules [46]. To the best of our knowledge, however, this has not been examined in primary MSCs and nondiseased articular chondrocytes. As opposed to chondrogenic differentiation, a few research groups have reported that chondrocytes may facilitate MSC osteogenic differentiation in coculture systems with [47] or without [48, 49] direct physical contact. This divergent outcome of chondrogenesis versus osteogenesis may result from the combined effects of changes in chondrocyte phenotype due to multiple expansions in monolayer prior to cocultivation [50, 51] and the use of high serum contents during the induction [52], both of which can interfere with differentiation mechanisms. In the present study, in order to better understand the role that primary chondrocytes play in directing MSC differentiation and to avoid the concerns addressed above, we developed a coculture system in which articular chondrocytes harvested from young calves were cultivated in the pellet format to maintain the phenotype and MSCs derived from the same groups of donors were kept separated from chondrocytes. Effects of serum and exogenous stimulators were eliminated in our system so that MSC differentiation mainly relied on paracrine signaling. In a pilot study directly comparing MSCs cultivated with serum-free, growth factor-free medium (IM) for 15 days with uncultured MSCs, no difference between the two populations was detected, suggesting that the phenotype of MSCs was maintained in the IM, although the IM did decrease the cell doubling rate compared with the EM (data not shown). Overall, our data reveal that MSCs in monolayer underwent a certain degree of chondrogenic differentiation under chondrocyte stimulation, and the level of differentiation was proportional to the number of chondrocytes present in coculture. Morphologically, differentiating MSCs tended to aggregate (Fig. 1C), which may result from elevated expression of SOX9 transcription factor in cells (Fig. 2C), which has been demonstrated to facilitate cell aggregation via upregulation of N-cadherin molecules [53, 54]. The increased expression of chondrogenic and hypertrophic markers in harvested monolayer cells with increasing chondrocyte number in coculture and the absence of osteogenic induction indicate a phenotype change in MSCs toward the chondrogenic lineage when MSCs are cultivated with and stimulated by chondrocytes. In addition, flow cytometry results (Fig. 3A) indicate that the proportion of CD166+ and CD44+ cells decreased with increasing chondrocyte/MSC ratio. This is supported by literature suggesting that differentiation can result in loss of surface antigens in MSCs in both mRNA [55] and protein [56, 57] levels. In the distribution diagram characterizing the size and granularity of harvested monolayer cells (Fig. 3B), the migration of MSC-differentiated cells from the MSC-main region (R1) to a new area (R2) implies that these cells were smaller and slightly smoother than MSCs and therefore became chondrocyte-like [13, 58]. Chiang et al. recently used atomic force microscopy to identify morphologies of chondrocytes and TGF-β-treated MSCs and found that MSCs stimulated by TGF-β resembled spherical chondrocytes, although the two cell types still had minor variations in their shape [58]. Moreover, we also found that cells harvested from the high chondrocyte content groups formed chondrocyte-like clusters (Fig. 4). This phenomenon is consistent with a study reported by Gelse et al. where MSCs infected with adenoviral vectors, either BMP-2 or IGF-1, formed clusters 4 days postinfection [59]. In order to assess the ability of differentiated cells to develop into neocartilage, we chose an initial chondrocyte/MSC ratio of 63:1 as our model for functionality experiments since elevated hypertrophic potential in the 100:1 chondrocyte/MSC group (Fig. 2F) is not preferred, and monolayer cells derived from this coculture group were named CDMD cells. Notably, in the first week of the tissue development, CDMD constructs had a compromised GAG content and equilibrium compressive modulus (Fig. 5), both of which are interrelated because negatively charged GAG molecules within native or engineered cartilage generate a repelling force against external compressive loading and thereby grant tissues basic stiffness. This suppressive effect is likely due to loss of CD44 molecules in newly differentiated cells (Fig. 3A). Rationally, CD44 is responsible for cell adhesion to hyaluronan, an extracellular macromolecule that provides binding sites for aggrecan monomers with GAG branches to attach [40]. Therefore, the disappearance of CD44 surrendered the binding of hyaluronan to cells, yielding reduced GAG encapsulated within constructs, and the decreased GAG content led to suppressed compressive moduli. We found that CDMD cells were able to recover the ability to express CD44 antigens during tissue development (Fig. 7), and the accumulated intensity of CD44 signals in both chondrocyte and CDMD groups increased over time as cells proliferated. At the end of the 4-week cultivation, CDMD cells exhibited a higher potential to develop into robust tissue-engineered cartilage with biomechanical and biochemical properties stronger than constructs grown from primary chondrocytes (Fig. 5A–5D). This outcome likely results from the combined influence of a higher proliferative rate (supplemental online Table 2) and continuously increased extracellular matrix (ECM) synthesis (Fig. 5E, 5F) of CDMD cells. Moreover, individual chondrocytes were shown to secrete more ALP molecules in the late stage of the tissue cultivation, whereas individual CDMD cells released a constant and relatively low level of ALP throughout the process (Fig. 6B). However, no calcium deposition was observed in either group by day 28 (Fig. 6C). This implies that the accumulated ALP amounts (Fig. 6A) were insufficient to calcify the structure in both cases and, more importantly, that CDMD cells were able to resist hypertrophy and calcification after long-term culture in vitro. Nevertheless, in vivo stability of CDMD products needs to be further investigated. In our coculture model, the production of CDMD cells that best support neocartilage development requires a chondrocyte/MSC ratio of 63:1. This level of chondrocytes can be practically obtained in cartilage tissue engineering strategies since several articular origins are available, such as a non-load-bearing portion of glenohumeral, elbow, and knee joints. Furthermore, allogeneic chondrocytes may be favored in this coculture application because a large number of cells can be isolated from younger donors without causing further damage to the diseased joint [60], and the in vitro induction minimizes the immune response of the host as opposed to in vivo processing. The short-term (approximately 2 weeks) coculture procedure also allows the reuse of the isolated chondrocytes within their limited life span since they are maintained in pellets that can be easily disassociated and reformed without disturbing the cell phenotype. And finally, engineered devices that simulate the gradual release of multiple identified paracrine factors by chondrocytes can be developed using novel techniques, such as layer-by-layer multilayer coating [17, 18], and substitute for chondrocytes as the inductive feeder. Conclusion The present work demonstrates for the first time that, in the absence of direct cell-cell contact and exogenous inducers, morphogenetic signals generated by healthy chondrocytes are sufficient to induce differentiation of bone marrow MSCs into a cell population that highly mimics articular chondrocytes in terms of their morphology, phenotype, and behavior. Our findings also show that although MSCs tend to lose the ability to express CD44, an important regulator in cartilage biology, during chondrogenic differentiation, CDMD cells can regain this function and develop into functional neocartilage that exhibits less hypertrophy. In our in vitro chondrocyte/MSC cocultivation model, we used juvenile articular chondrocytes in a clinically relevant serum-free culture environment. The juvenile chondrocytes are preferred because of their stronger potential for ECM formation and molecule release and a release profile that differs from that of aged or diseased chondrocytes [61]. Our model enables the emulation and decomposition of in vivo conditions in order to understand paracrine regulation of articular chondrocytes and bone marrow MSCs in MSC differentiation during skeletal development. Identification of the specific individual or combined effects of chondrocyte-secreted paracrine signals on MSC chondrogenesis requires further investigation. The current study provides a basis for improved protocols to direct MSC differentiation toward the development of cartilage tissue replacements suitable for implantation. Acknowledgments This work was supported by the National Science Foundation (NSF0602608). 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TI - Coculture-Driven Mesenchymal Stem Cell-Differentiated Articular Chondrocyte-Like Cells Support Neocartilage Development
JF - Stem Cells Translational Medicine
DO - 10.5966/sctm.2012-0083
DA - 2012-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/coculture-driven-mesenchymal-stem-cell-differentiated-articular-WIzC593tNI
SP - 843
EP - 854
VL - 1
IS - 11
DP - DeepDyve
ER -