Enhanced Chondrogenesis in Co-Cultures with Articular Chondrocytes and Mesenchymal Stem Cells

2.1. Scaffold preparation

Nonwoven poly(ε-caprolactone) (PCL) microfiber mats were fabricated using a horizontal electrospinning setup with a copper ring to stabilize the electric field as previously described [17]. Mats were electrospun to an average fiber diameter of approximately 10 μm (9.6 ± 2.0 μm) using a solution of 18 wt % PCL (Inherent viscosity range 1.0 – 1.3; Durect corporation, Cupertino, CA) in a 5:1 volume ratio of chloroform to methanol. The polymer solution was pumped at a flow rate of 40 mL/h through a blunt 16 G needle, while charged with an applied voltage of 30 kV to draw microfibers toward the collector plate 33 cm from the needle tip. The resulting PCL mats of approximately 1.3 mm thickness were aerated, inspected for consistent microfiber morphology using SEM, and stored in a desiccator.

PCL scaffolds were prepared by die-punching 8-mm-diameter discs from the electrospun mats using a dermal biopsy punch. The scaffolds were then sterilized by exposure to ethylene oxide (AN74i, Andersen Sterilizers, Haw River, NC) at ambient temperature for 14 h and aerated overnight to remove residual fumes. In order to eliminate air bubbles and to improve cell adhesion, scaffolds were prewetted by centrifugaton through a graded series of ethanol (100 % – 20 %), followed by two rinses in phosphate buffered saline (PBS), and incubation in general culture medium (high-glucose DMEM, 10 % fetal bovine serum (FBS), penicillin/streptomycin/fungizone (PSF)) for three days.

2.2. Cell harvest and expansion

Bovine MSCs and ACs were harvested from 7-10 day old calves (Research 87, Boylston, MA), less than 36 h after slaughter using previously established protocols [18]. Briefly, bone marrow was harvested from tibiae and femora, diluted with culture medium and separated from fat by centrifugation. Marrow isolations were plated onto culture flasks and allowed to adhere for 48 h before washing with PBS to remove non-adherent cells and blood clots. Cultures were maintained in general medium until confluent and passaged for sub-cultures. After two passages, MSCs from a minimum of three animals were pooled, aliquoted, and frozen in freezing medium (DMEM with 20 % FBS and 10 % dimethyl sulfoxide). Articular cartilage (from the same animals) was collected from the femoral condyle area, minced to approximately 1x1x1 mm, washed with PBS and digested in chondrocyte culture medium (DMEM, 10% FBS, 1% non-essential amino acids, 50 μg / mL ascorbic acid, 46 μg / mL L-proline, 20 mM HEPES, PSF) containing 2 mg / mL collagenase type II (Worthington biochemical corporation, Lakewood, NJ) while incubating at 37 °C overnight. Primary ACs were pooled, aliquoted, and frozen in freezing medium.

Rabbit MSCs were harvested and isolated as previously described [19]. Briefly, bone marrow was aspirated from the tibiae of six 6 month old male New Zealand white rabbits (Myrtle's Rabbitry Inc., Thompsons Sta, TN). Under general anesthesia, bone marrow from each leg was aspirated into a 10 mL syringe containing 5000 U / mL heparin to prevent coagulation. The bone marrow was plated in tissue culture flasks and non-adherent cells were washed away after 72 h. Cultures were maintained in general medium until confluent, cells from all animals were pooled together, aliquoted, and frozen in freezing medium.

2.3. Cell seeding and chondrogenic culture

Two independent cell cultures were performed: one with primary (p⁰) bovine ACs and (p³) bovine MSCs, one with culture expanded (p²) bovine ACs and (p²) rabbit MSCs. The purpose of the xenogeneic model was to facilitate the cell type specific gene expression analysis of co-cultured chondrocytes and MSCs. In both cases, scaffolds were seeded with pure AC and MSC populations as well as their combinations, with 1:1 and/or 1:3 AC-to-MSC ratios.

The frozen AC and MSC aliquots were thawed and sub-cultured as indicated to establish seeding suspensions of 1.25×10⁶ cells / mL. In preparation for cell seeding, prewetted scaffolds were press-fitted into cylindrical poly(methyl methacrylate) cassettes designed to confine the seeding suspension. Cells (in ~200 μL of general medium) were then pipetted onto each scaffold to achieve a final seeding density of 4×10⁶ cells / mL scaffold volume. Cells were allowed to adhere for 4 h before culture medium was gently added to completely cover the cassettes. Scaffolds were removed from the cassettes after 24 h and placed in 12 well culture plates with 4 mL of serum free chondrogenic medium (high-glucose DMEM, 1 % ITS+ premix [BD Biosciences, San Jose, CA], 50 μg / mL ascorbic acid, 100 nM dexamethasone, 10 ng / mL TGF-beta 3 [PeproTech, Rocky Hill, NJ], PSF). Half of the medium was replenished two to three times a week. Four replicate scaffolds were harvested after 0, 14, and 28 days of chondrogenic culture and washed with PBS. A 3 mm biopsy punch was then used to obtain individual samples for each analysis.

2.4. Biochemical assays

Two 3 mm biopsy samples were pooled together from each scaffold and frozen in -20 °C until used for biochemical assays. Thawed samples were digested in 500 μL of a proteinase K solution (1 mg / mL proteinase K, 0.01 mg / mL pepstatin A and 0.185 mg / mL iodoacetamide in a 50 mM tris(hydroxymethyl aminomethane) – 1 mM ethylenediaminetetraacetic acid buffer, pH 7.6) in a 56 °C water bath for 16 h. DNA and matrix components were extracted via two additional freeze-thaw cycles followed by 10 min sonication.

DNA content of the scaffolds was determined using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Eugene, OR). Briefly, cell lysate, assay buffer and dye solution were combined in an opaque 96-well plate in duplicates, and allowed to incubate for 10 min at room temperature. Fluorescence was measured using excitation and emission wavelengths of 485 nm and 528 nm (FL x800 Fluorescence Microplate Reader; BioTek Instruments, Winooski, VT), respectively, and DNA concentrations were determined relative to a lambda DNA standard curve.

Glycosaminoglycan (GAG) content was determined using the colorimetric dimethylmethylene blue assay [20]. Briefly, cell lysate and color reagent were combined in a transparent 96-well plate in duplicates, and allowed to incubate for 7 min at room temperature. Absorbance at 520 nm was measured (PowerWave x340 Microplate Reader; BioTek Instruments), and GAG concentrations were determined relative to a chondroitin sulfate standard curve. For glycosaminoglycan synthetic activity, the resulting GAG amounts were normalized to the amount of DNA for each sample.

2.5. Histology

One 3 mm biopsy sample from each scaffold was fixed for histology in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA), then immersed in 70% ethanol prior to embedding in HistoPrep freezing medium (Fisher Scientific). Frozen sections 5 μm thick were cut using a cryostat (Leica CM 1850 UV; Leica Biosystems Nussloch GmbH, Germany), mounted onto glass slides, and placed on a 42 °C slide warmer to facilitate adhesion. Sections were stained with Safranin O to visualize the distribution of cartilaginous extracellular matrix. Images were obtained using a light microscope with a digital camera attachment (Axio Imager.Z2 equipped with AxioCam MRc5; Carl Zeiss MicroImaging GmbH, Germany).

2.6. Real-time reverse transcription polymerase chain reaction

After 3 mm samples of the scaffolds were removed for biochemical analysis and histology, the remainder of the scaffolds were minced to approximately 1×1×1 mm pieces and used for RNA isolation using an RNeasy mini kit (Qiagen, Valencia, CA). Briefly, minced scaffolds were placed into 600 μL of lysis buffer and incubated at room temperature for 30 min with periodical vortexing. Cell lysate was then passed through a QIAshredder homogenization column and stored at -80 °C until further processing. An equal volume of 70 % ethanol was added to thawed lysates and RNA isolation was continued following the animal cell protocol provided by the manufacturer. Reverse transcription was then carried out to synthesize cDNA from purified RNA samples using Oligo(dT) primers (Promega, San Luis Obispo, CA) and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Finally, cDNA was subjected to real-time PCR (Applied Biosystems 7300 Real-Time PCR System, Foster City, CA) using SYBR Green detection (PerfeCTa SYBR Green FastMix, ROX; Quanta Biosciences, Gaithersburg, MD) with custom designed primers (Integrated DNA Technologies, Coralville, IA).

Primer sequences are given in Table 1. Species specificity (>99%) and PCR efficiency (>95%) of each primer pair was verified before use. Target gene expression was first normalized to the expression of the housekeeping gene GAPDH in the same sample (ΔCt), then to the average baseline expression of that target gene measured in the bovine AC group at the start of chondrogenic cultures (ΔΔCt). Finally, the 2^-ΔΔCt method was used to convert normalized gene expression levels to fold differences and statistics were calculated on these values [21]. Similarly, 2^-ΔCt was used to calculate the ratios of collagen II / collagen I and bovine-specific / cross-species expression within individual samples.

2.7. Statistics

Results are presented as means ± standard deviations. Statistical analysis was performed with an SPSS 16.0 software package (SPSS, Chicago, IL). Biochemical assay data were analyzed using one-way ANOVA followed by Tukey's post-hoc test, whereas RT-PCR data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test. Differences were considered significant at 95 % confidence level.

3.1. Scaffold cellularity

A continuous increase in scaffold cellularity was observed in all culture groups with primary bovine ACs and bovine MSCs, as seen in Figure 1A. The presence of primary chondrocytes enhanced cell proliferation as the DNA content in the beginning of the cultures was the highest but at later times the lowest with pure MSCs. Similarly, increasing cellularity was observed also with passaged bovine chondrocytes whereas DNA content in the pure rabbit MSC group decreased in culture (Figure 1B). Furthermore, there were no differences between primary AC and co-culture groups at any time, whereas the passaged AC group had significantly lower DNA contents in comparison to the corresponding co-culture group.

3.2. GAG synthesis

All cultures with primary bovine ACs and bovine MSCs showed continuously increasing GAG contents and synthetic activity, as seen in Figures 2A and 2B. In the beginning of the chondrogenic cultures, GAG synthesis was strongest with the primary AC group, whereas at later times co-cultures reached similar or even higher levels of GAG content and synthesis than pure ACs. With passaged bovine ACs and their co-cultures, GAG contents and synthesis increased during the first two weeks, but then remained at similar levels for the remainder of the cultures (Figure 2C and 2D). The increase was larger with co-cultures than with pure ACs. Rabbit MSCs showed relatively high synthetic activity in the early cultures, whereas GAG contents actually decreased during culture (due to low cellularity).

3.3. Histology

Histological analysis showed that cells and extracellular matrix (ECM) accumulated mostly on the upper part of the scaffolds, with increasing cellular infiltration during the culture. Histology corroborated the results of GAG measurements. Primary ACs and their co-cultures had similar histological appearances, whereas clearly thinner cell/ECM crust was observed in pure bovine MSC cultures (Figure 3A). In contrast to primary cells, co-cultures with passaged ACs produced much higher tissue contents than ACs alone (Figure 3B). Furthermore, only very limited amounts of cells/ECM were observed with pure rabbit MSCs.

3.4. RT-PCR

RT-PCR analysis was performed only with passaged ACs and rabbit MSCs. As expected, the initial gene expression levels for aggrecan and collagen types I and II were lowest with undifferentiated MSCs and highest with pure chondrocytes, and the same was true also for the collagen type II-to-type I expression ratio (Figure 4). MSC and co-culture groups showed strong chondrogenic induction already after two weeks of culture, and after four weeks they had similar or even higher gene expression levels than pure chondrocytes. Chondrogenic markers were always higher in co-culture than in the MSC group, except that the highest aggrecan expression after four weeks was seen in MSCs.

Species-specific analysis of gene expression showed a strong continuous decrease in the proportion of rabbit MSCs in the co-cultures. In the beginning, the majority of GAPDH and collagen I signal came from rabbit MSCs, whereas collagen II and aggrecan expression were predominantly of bovine origin (Figure 5). This was consistent with the relative expression levels in pure AC and MSC populations combined with the 1:3 AC-to-MSC seeding ratio. The situation had changed already after two weeks and all studied markers indicated a mostly bovine cell population, with further increase in bovine specific signal at the end of the culture period.