Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

Isolation and characterization of human bone marrow stromal cells

Human bone marrow samples were obtained from healthy adults (male, age =21 + 2.6 years) who were undergoing fracture fixation involving the long bones. After providing the patient information sheet and explaining the patients on bone marrow collection, written informed consent was obtained and bone marrow was collected. This study was approved by the University of Malaya Medical Center Ethics Committee (reference no. 602.22). Human bone marrow was collected (N = 6) in sterile 3-ml BD Vacutainer blood tubes (K2 EDTA, BD, Franklin Lakes, NJ, USA) by orthopedic surgeons and was kept at 4 °C until isolation. The mononuclear cells were isolated using Ficoll density gradient method as described earlier (Krishnamurithy et al., 2014). Isolated cells were cultured and expanded until P3 in T-75 flasks. The isolated cells were characterized as MSCs through flow cytometry using cell surface markers (positive and negative markers) and its ability to undergo trilineage differentiation (adipogenic, chondrogenic, and osteogenic differentiation) was described earlier (Tan et al., 2013). MSCs isolated form three biological samples were used for gene expression analysis and three biological samples were used for imaging analysis.

Chondrogenic differentiation and experimental groups

Pellet culture: MSCs were harvested at P3, and 2.5 × 10⁵ cells were pelleted in a 15-ml propylene centrifuge tube at relative centrifugal force (RCF) of 260 × g 5 min. After removal of the supernatant, the pellet cultured with 2 ml of chondrogenic medium contains the following: DMEM high glucose (4.5 mg/ml d-glucose) with sodium pyruvate (110 µg/ml) (Invitrogen, Carlsbad, CA, USA), 50 mg/ml ITS (Sigma) (1×) (Invitrogen), l-ascorbate 2 phosphate (50 µg/ml) (Sigma, St. Louis, MO, USA), 10 ng/ml TGFβ3 (Invitrogen), 100 nM dexamethasone (1 × 10⁻⁷ M) (Sigma), 100 µg/ml penicillin/streptomycin (Invitrogen), and 40 µg/ml l-proline (Sigma). The media were changed every 3 days.

Alginate cell constructs: 1.2% alginate prepared from alginic acid powder, low viscosity (Sigma-Aldrich) in 0.9% sodium chloride (NaCl) and filtered sterile by a 0.2-µm filter. MSCs at P3 were harvested, and a concentration of 4 × 10⁶ cells per milliliter of alginate was obtained before dropping into sterile calcium chloride solution (CaCl₂) using a pipette. Alginate bead constructs were cross-linked in this solution for 10 min in an incubator at 37 °C, were rinsed in 0.9% normal saline 2–3 times, and then transferred to the culture dishes (ultra-low attachment 6-well plates; Corning, New York, NY, USA). Three beads were placed per well (about 80,000 cells per bead) and supplemented with 2 ml of chondrogenic medium. The medium was changed every 3 days. Alginate cell constructs were dissociated in a buffer solution containing 0.015 M sodium citrate and 0.15 M sodium chloride (Na₃C₆H₅O₇ dehydrate 2H₂O, MW = 294.10), pH 7.2, and centrifuged at 1,100 rpm for 5 min before gene expression studies were conducted.

Monolayer culture: MSCs at P3 were cultured in chondrogenic medium at a density of 4,000 cells per centimeter in 6-well plates.

Glycosaminoglycan (GAG) analysis and DNA quantification

Alginate beads and pellets from days 3, 12, and 21 were washed with phosphate-buffered saline. Alginate bead was dissolved in sodium citrate and the pellets were crushed and the samples were digested in papain buffer overnight at 60 °C. The digested samples were subjected to biochemical analyses to determine the glycosaminoglycan and DNA content. GAG production was determined by using Blyscan assay kit (Biocolor, arrickfergus, Northern Ireland), according the manufacturer’s protocol. GAG content was determined using a standard curve drawn using standard solutions containing chondroitin 4-sulfate. Alginate beads without cells at the respective time point was analyzed in the same manner and the values are used as blank. DNA content was determined by using the fluorescent picoGreen dsDNA quantification assay (Invitrogen). GAG in alginate bead and pellet culture system was expressed as µg GAG per µg of DNA.

Morphological studies

On day 21, samples were fixed in 10% formalin and processed for histological studies stained with Safranin O Fast green or immunohistochemistry for Collagen type II (Mouse monoclonal anti-Human; Merk, Darmstad, Germany) in 1/100 dilution and aggrecan (Mouse monoclonal anti-Human; Abcam, Cambridge, UK) in 1/50 dilution using EnVision+ System-HRP (DAB) Dako kit (Dakocytomation, Glostrup, Denmark) according to the manufacturer’s protocol.

RNA isolation and cDNA synthesis

Total RNA was isolated from cultures of pellet, alginate, and monolayer on days 3, 12, and 21. RNA was isolated using SV total RNA isolation system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. One hundred nanograms of RNA was used to generate cDNA with iScriptTM Reversed Transcription Supermix for RT-qPCR (BioRad) according to the manufacturer’s protocol.

Quantitative real-time polymerase chain reaction

PCRs were carried out in duplicate for each three biological sample in a final volume of 20 µl containing SYBR Green mastermix (BioRad, Hercules, CA, USA) with 160 nM concentration of each primer and 100 ng cDNA in 0.2-ml PCR tubes using CSFX96TM Real Time System, Bio-Rad, under the following conditions: 3 min at 95 °C followed by 40 cycles at 58.6 °C for 0.20 s as annealing temperature and 72 °C for 0.30 s as extension. The reactions were ended by 0.1 min at 95 °C and a melt curve by increasing temperature from 65 °C, 0.05 min, to 95 °C, 0.5 min, stepwise. No template controls were used in each reaction as negative control. The data were presented as a time fold change relative to the internal control gene expression. The data were then normalized to transcription levels of day 0 culture using ΔCT and ΔΔCT methods. Values below 1 were considered downregulated. The following primer sets were applied in this experiment (Table 1).

Transmission electron microscopy (TEM)

Samples for TEM study were fixed with 4% glutaraldehyde in cacodylate buffer for 24 h at 4 °C and postfixed in buffered 1% osmium tetra oxide for 2 h at 4 °C, washed with cacodylate buffer 2–3 times, and kept overnight at 4 °C in cacodylate buffer before en bloc staining with 4% uranyl acetate (Agar Scientific Ltd R1043, Essex, England) in double distilled water for 10 min. Samples were then dehydrated in ascending series of ethanol and embedded in Epon (Agar 100 resin kit; Agar Scientific Ltd R1043).

Semi-thin 1 µm sections were obtained using a Leica ultramicrotome (Reichert Ultracuts; Leica Microscystem, Vienna, Austria) and stained with toluidine blue for 1–2 min before viewing with light microscopy. Ultrathin sections were cut at 70–80 nm, collected on copper grids (300 meshes), and stained with uranyl acetate and lead citrate (Agar Scientific). Images were viewed using TEM (Leo Libra 120; Carl Zeiss SMT AG, Oberkochen, Germany).

Statistical analysis

The difference between experimental groups was calculated using nonparametric Kruskal-Wallis H test and the difference between two independent experimental groups using Mann–Whitney U test, available on the statistical software package SPSS (version 18.0), with p ≤ 0.05 being considered significant.

Chondrogenic differentiation

Chondrogenic differentiation of MSCs in different experimental groups was shown in Fig. 1. Safranin O Fast green staining of pellet culture (Fig. 1A) on day 21 showed red staining positive for GAG and green area indicating non-GAG depositions. In alginate culture on day 21, Safranin O Fast green showed intense staining of red color (Fig. 1B), indicating high GAG production in this group compared with control group (Fig. 1C).

Immunohistochemistry for Collagen type II and aggrecan

Monolayer, pellet, and alginate bead cultures showed positive for Collagen type II staining on 21 days of culture (Figs. 2A–2C), compared with the negative controls (Figs. 2G–2I). Immunochemistry of aggrecan showed positive for pellet and alginate bead cultures (Figs. 2D–2E); however, no positive staining was observed in monolayer (Fig. 2F) compared with the negative control (Figs. 2J–2L).

Glycosaminoglycan/DNA quantification

Figure 3 represents the GAG/DNA content of alginate bead and pellet cultures. The results indicate that hMSCs differentiated in alginate beads has produced significantly higher (p < 0.05) glycosaminoglycan than cells in the pellet culture system. The increase in GAG content was also significant over time, day 3 to day 12 and day 12 to day 21, in both alginate bead and pellet culture.

Chondrogenic gene expression

Expressions of chondrogenic genes, Collagen II, Sox 9, and aggrecan, were shown in Figs. 4A–4C, respectively. In all time points (days 3, 12, and 21), alginate culture represents significantly higher (p < 0.05) chondrogenic gene expression compared with monolayer and pellet cultures. In alginate group, there was a significant increase between days 3 and 12 and no significant increase was observed between day 12 and 21 for Collagen II and Sox 9, whereas no significant increase was observed between days 12 and 21 for aggrecan. For pellet culture, there was a significant increase from days 3 to 21 for Collagen II and Sox 9 and expression of Sox 9 and aggrecan remains non-significant from day 12 to 21.

Hypertrophic gene expression

The expressions of hypertrophic genes Collagen X and Runx2 are shown in Figs. 5A and 5B, respectively. The expressions of these genes were significantly higher (p < 0.05) in alginate beads at the earlier time point (day 3) compared with pellet culture; on the other hand, these genes were found to be significantly down regulated in the later time points of days 12 and 21. At day 3 no significant difference was observed between the expression of these genes between monolayer and alginate bead culture. In pellet culture, although the expressions of hypertrophic genes were significantly lower on day 3, the expression level was significantly increased over time.

Cell adhesion molecule expression

N-CAM1 and N-cadherin (Figs. 6A and 6B) remained downregulated on days 3 and 12 in pellet culture and upregulated on day 21, but in alginate group, N-CAM1 was upregulated over time from days 3 to 21, and for alginate and pellet cultures, N-cadherin remained downregulated on days 3 and 12 but is upregulated on day 21. In monolayer, the expression of N-CAM1 was not significant on days 3 and 12, whereas it was seen upregulated on day 21 and N-cadherin was increased significantly over time.

TEM of MSCs in pellet and alginate cultures

Resin-embedded samples of pellet and alginate cultures on day 21 and MSCs on day 0 were cut into semithin 1-µm sections, stained with toluidine blue, and studied under light microscopy (Fig. 7). Ultrathin sections of 70 nm thickness on copper grids were cut, stained with uranyl acetate and lead citrate, and studied with electron microscopy (Figs. 8 and 15).

The prominent features of semithin sections of alginate beads compared with pellet culture were lower cellularity and higher intercellular spaces (Fig. 7B). In alginate culture, cells were arranged in small groups of two or three cells per group and the matrix between them was stained purple using toluidine blue (Fig. 7B), whereas in pellet culture, cells were in closer contact with each other and less purple color indicated lower extracellular deposition (Fig. 7C).

The presence of abundance of polyanions in the ECM of cartilage gives a purple color to the metachromatic dye such as toluidine blue. Samples of MSCs on day 0 as a control group stained blue, whereas samples of chondrogenic differnetiated mesenchymal stromal cells (CMSCs) in alginate and pellet cultures showed positive metachromatic areas (purple color). However, no difference was observed between alginate and pellet culture and this might be due to the limitation of ultra-thin sections used.

Ultrathin sections of pellet and alginate beads containing chondrogenic MSCs

In monolayer, MSC surface formed filopodia or blebs associated with dense bodies (Figs. 8B and 8C) and cytoplasm occupied with rER, free ribosomes, mitochondria (M), and vacuoles (Fig. 8). Nuclei contains euchromatin, usually with a distinct nucleolus, demonstrating an active protein-synthesizing cell; in some cells, the nucleus was convoluted (Fricker et al., 1997) (Fig. 8A).

In alginate bead culture, ultrastructure of nucleus in CMSC showed euchromatin with a variety of phenotypes, round, oval, or slightly indented (Figs. 9 and 12). Chondrogenic alginate shows active protein synthesis, with euchromatic nuclei, prominent nucleoli, and abundant rER filled with electron lucent materials producing abundant ECM, containing collagen fibers (Figs. 9, 10 and 12) and molecules with side branches similar to proteoglycan aggregates among fibers (Fig. 10B). Chondrogenic differentiated cells are arranged in small groups as they were seen in lower magnification in semithin (Fig. 7B) and ultrathin sections (Figs. 9B and 12A). Higher magnification of adjacent cells did not show any junctional complexes between the cells. Smaller vesicles contained electron lucent material near cell membrane (Fig. 12A) that might originate from rER releasing their content to ECM, causing the cells grow apart from each other, similar to that seen during the interstitial growth in cartilage. The ECM was rich in collagen fibers with banding patterns (Fig. 10). In active protein synthesis, chondrogenic cells in alginate were difficult to distinguish between distended rER and Golgi apparatus (Fig. 9A); however, in higher magnification, the rER can be easily recognized with studded ribosomes (Fig. 11B, black arrow).

Two subpopulations of cells can be distinguished in ultrathin sections of pellet culture; In one group, signs of cell death appeared with abundant cytoplasmic vesicles, lipid droplets, free ribosomes, swelled or fused mitochondria (Fig. 13A), vacuoles, expelling of cytoplasmic organelles to ECM (Fig. 13B), indented nucleus (Fig. 13D), or cells without a prominent nucleus (Fig. 13C). The second population consisted of active protein-synthesizing cells with a euchromatin and round nucleus and abundance of collagen fibers secreted in the ECM (Figs. 14 and 15). Golgi apparatus can be observed in both groups (Figs. 13D and 14A) as machinery for synthesis of carbohydrates (Alberts et al., 2002) or GAGs in chondrogenic-induced MSCs.