Cartilage fragments from nasal septum were obtained from 15 donors between 25 and 60 years old, which underwent septoplasty surgery procedures, after informed consent and approval of the Ethics Committee of the University of Ferrara and S. Anna Hospital. Briefly, cartilage fragments were minced into small pieces and rapidly incubated with type VIII Collagenase (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 37°C for 16 h (do Amaral et al., 2012). Cells were harvested by centrifugation and plated (p0) at a density of 20,000 cells/cm² in tissue culture flasks (25 cm²) or 8-well culture slides in standard medium (50% DMEM high-glucose/50% DMEM F-12/10% fetal calf serum) (Euroclone S.p.A., Milan, Italy) supplemented with antibiotics (penicillin 100 mg/mL and streptomycin 10 mg/mL), at 37°C in a humidified atmosphere of 5% CO₂. After 7 days, the culture medium was removed and then changed twice a week. At 70–80% confluence, cells were scraped off by 0.05% TRYPSIN EDTA (Gibco, Grand Island, NE, USA), washed, plated, and allowed to proliferate in standard conditions (50% DMEM high-glucose/50% DMEM F-12/10% fetal calf serum) to induce chondrocyte dedifferentiation (until p6). Dedifferentiated chondrocytes at different culture passages were scraped off counted by hemocytometric analysis, assayed for viability, and thereafter used for molecular analysis or for encapsulation procedures. During the in vitro expansion, dedifferentiated chondrocytes underwent a total of 19–21 population doublings.

Porcine urinary bladders were harvested from pigs, immediately following euthanasia. The connective tissue excess and the residual urine were removed. By mechanical treatment, the tunica serosa, tunica muscularis externa, tunica submucosa, and majority of the tunica muscularis mucosa were removed; thereafter, urothelial cells of the tunica mucosa were detached from the luminal surface by incubating the tissue in saline solution. The resulting tissue, which was composed of the basement membrane of the urothelial cells plus lamina propria, is termed “urinary bladder matrix,” shortly UBM. The obtained UBM sheets were then treated by a solution containing 0.1% (v/v) peracetic acid (Sigma), 4.0% (v/v) ethanol (Sigma), and 95.9% (v/v) sterile water for 2 h. Peracetic acid residues were then removed with washes with phosphate-buffered saline (PBS, pH 7.4) and sterile water. The decellularized UBM was then lyophilized and milled to obtain a particulate form using a Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ, USA).

Fluidic reagents were introduced into the microfluidic network from glass gastight syringes (Hamilton, Reno, NV, USA) by syringe pumps (model KD100, KD scientific Inc., Holliston, MA, USA).

“Empty” and “multifunctional” (containing cells, ECM components or both) barium alginate microfibers were produced with a snake micromixing microfluidic device (Figure 1A). A sodium alginate dispersion in water (1.5–2.5%, w/v), used as main constituent of the microfibers was introduced into one inlet of the microchip at appropriate flow rates (0.50–1.50 mL/min). Plain sodium alginate dispersion or sodium alginate suspensions were delivered via the second inlet. The suspensions contained different amounts (1.125–4.500% for gelatin or 0.1–1.0% for UBM) of either ECM components (i.e., gelatin or UBM) or cells (i.e., dedifferentiated chondrocytes at 2 × 10⁶ cells/mL). Notably, gelatin forms a homogeneous dispersion in the aqueous sodium alginate, whereas UBM, being unsoluble in water, forms a suspension. The output from the outlet of the chip was transferred via a Teflon tube, into a BaCl₂ solution (6.0 mM in water) where the Na-alginate flow stream was gelled to produce the final Ba-alginate consolidated microfibers. The plain alginate, the alginate/gelatin, or alginate/UBM microfibers are named Af, AGf, and AUBMf, respectively. Cells-containing microfibers were washed three times with saline before culturing in standard chondrocyte medium at 37°C in a humidified atmosphere of 5% CO₂.

The dimension and morphology of microfibers were evaluated by optical stereomicroscopy. Quantitative analyses were performed using the photomicrograph analysis software EclipseNet. The mean diameter of the microfibers (±SD) was obtained by taking nine measurements along the (10 mm) length of the samples at equal intervals, in triplicate. Additionally, the distribution of different amount of gelatin (1.125, 2.25, and 4.5% w/v) and UBM (0.1, 0.5, and 1% w/v) in composite microfibers was evaluated by Coomassie Brilliant Blue staining. For the staining, the microfibers were incubated in 0.1% Coomassie Brilliant Blue R-250, 50% methanol, and 10% glacial acetic acid at room temperature for 1 h and then washed overnight at room temperature in the destaining solution (40% methanol and 10% glacial acetic acid). Gelatin and UBM distribution was determined by optical stereomicroscopy.

Dedifferentiated chondrocytes from monolayer culture were harvested and resuspended in standard medium at 2 × 10⁷ cells/mL density. Droplets (10 µL) were carefully placed in a 12-well plate. Cells were allowed to adhere for 3 h, followed by the addition of 1 mL of standard medium supplemented with 10% FCS. After 24 h, the cell droplets coalesced and became spherical. Medium was changed every 3 days, and micromasses were harvested on days 7 and 14.

Viability of encapsulated cells was assessed immediately after encapsulation procedure and at days 7 and 14 of culture. Cell viability was evaluated by Calcein-AM/propidium iodide (PI) staining (Cellstain double staining kit, Sigma-Aldrich).

Before staining, the medium was removed from the wells, and 500 µL of the staining solution was added to each well. The samples were incubated in the dark at room temperature for 15 min, thereafter the wells were rinsed with PBS and immediately imaged using epifluorescent microscopy. Viability was measured via cell counting and automated analysis using ImageJ. Samples were visualized under a fluorescence microscope (Nikon, Optiphot-2; Nikon Corporation, Tokyo, Japan) dead cells stained red, while viable ones appeared green (Penolazzi et al., 2012).

The cells embedded in microfibrous scaffolds were retrieved, after gel dissolution at days 7 and 14 of in vitro culture. Microfibers were dissolved by incubation at 37°C in a 100 mM EDTA solution (pH 7.0) for 10 min. Cell suspension after microfiber dissolution was filtered with a Falcon™ 70 µM Nylon Cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) and employed for successive analyses.

Dedifferentiated chondrocytes before encapsulation procedure or recovered chondrocytes from microfibers or micromasses were lysed with 50 µL of RIPA buffer. Total sulfated GAG content was determined in RIPA samples from days 7 and 14 cultures by using 1,9-dimethylmethylene blue (DMMB), as previously described (Caron et al., 2012). A standard curve of chondroitin sulfate in PBS-EDTA was included to determine the GAG concentration in the samples. About 100 µL of diluted RIPA sample (5 µL RIPA sample and 95 µL PBS-EDTA) or standard (95 µL standard and 5 µL RIPA) was added to 200 µL of DMMB solution, and the extinctions were determined spectrophotometrically at 595 nm. GAG content was determined using the generated standard curve corrected for DNA content and expressed as µg GAG/ng DNA. DNA content in the same RIPA samples was determined using SYBR^® Green I Nucleic Acid stain (Invitrogen). A serially diluted standard curve of genomic control DNA (DNA Ladder G571A, Promega) in TE buffer was included to quantify DNA concentration in the samples. Before measurement, RIPA samples were diluted in TE buffer (1 µL RIPA sample and 99 µL TE buffer) and standard were prepared (99 µL standard in TE and 1 µL RIPA buffer). SYBR^® Green was diluted 10,000 times in TE buffer, and 100 µL of this solution was added to 100 µL of the above prepared samples or standards. Fluorescence was determined in standard 96-well plates in a SpectraFluor Plus reader (Tecan): excitation 485 nm and emission 535 nm. DNA concentration was determined using the standard curve.

Total RNA was extracted from dedifferentiated chondrocytes before encapsulation procedure, encapsulated chondrocytes cultured in alginate-based scaffolds, or the same cells maintained in micromass by using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. Total RNA was used for reverse transcription and stored at −80°C. Briefly, cDNA was synthesized from total RNA (500 ng) in a 20-µL reaction volume using the TaqMan High-Capacity cDNA Reverse Transcription kit (Applied Biosystems), as previously described (Lolli et al., 2014).

Gene expression analyses by RT-qPCR were performed for Col1A1, Col2A1, Aggrecan, and Col10A1 mRNA levels. The following Taqman probes were used: Col1A1 5′FAM-AAGACGAAGACATCCCACCAATCAC-NFQ3′, Col2A1 5′FAM-CCTGGTCTTGGTGGAAACTTTGCTG-NFQ3′, Aggrecan 5′FAM-CGCTGCCAGGGATCCTTCCTACTTG-NFQ3′, Col10A1 5′FAM-ATAAAGAGTAAAGGTATAGCAGTAA-NFQ3′ (Life Technologies, Carlsbad, CA, USA).

The CFX96™ PCR detection system (Bio-Rad, Hercules, CA, USA) was used, and results were calculated using the 2^−ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase as reference gene for normalization.

Glycosaminoglycan content was assessed by Alcian Blue staining in monolayered cells. Cells were rinsed with PBS and fixed in 10% formaldehyde in PBS for 10 min. Cultures were then stained with Alcian Blue pH 2 (1% in 3% acetic acid) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) 30 min at 37°C. Subsequently, cells were washed with water and observed using a Leitz microscope. The presence of GAG deposits appeared as blue staining areas.

Immunocytochemistry analysis was performed on freshly, dedifferentiated, and recovered chondrocytes employing the ImmPRESS (Vectorlabs, Burlingame, CA, USA) or 4plus AP universal (Biocare Medical, Concord, CA, USA) detection kits. Cells grown in chamber slides were fixed in cold 100% methanol and permeabilized with 0.2% (v/v) Triton X-100 (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) in TBS (Tris-buffered saline). Cells were treated with 3% H₂O₂ in TBS and incubated in 2% normal horse serum (Vectorlabs, Burlingame, CA, USA) for 15 min at room temperature. After the incubation in blocking serum, the different primary antibodies were added and incubated at 4°C overnight: polyclonal antibodies for Col1A1 (rabbit anti-human, 1:200 dilution, Santa Cruz Biotechnology, CA, USA), Col2A1 (mouse anti-human, 1:200 dilution, Abcam, Cambridge, UK), Aggrecan (mouse anti-human, 1:200 dilution, Santa Cruz Biotechnology, CA, USA), and Sox9 (rabbit anti-human, Santa Cruz Biotechnology, CA, USA). Cells were then incubated in Vecstain ABC (Vectorlabs, Burlingame, CA, USA) or Universal AP detection (Biocare Medical, Concord, CA, USA) reagents for 30 min and stained, respectively, with DAB solution (Vectorlabs, Burlingame, CA, USA) or Vulcan Fast Red chromogen kit (Biocare Medical, Concord, CA, USA). After washing, cells were mounted in glycerol/TBS 9:1 and observed using a Leitz microscope. Quantitative image analysis of immunostained cells was obtained by a computerized video-camera-based image-analysis system (with NIH, USA ImageJ software, public domain available at: http://rsb.info.nih.gov/nih-image/) under brightfield microscopy. Briefly, images were grabbed with single stain, without carrying out nuclear counterstaining with hematoxylin, and unaltered TIFF images were digitized and converted to black and white picture to evaluate the distribution of relative gray values (i.e., number of pixels in the image as a function of gray value 0–256), which reflected chromogen stain intensity. Images were then segmented using a consistent arbitrary threshold 50% to avoid a floor or ceiling effect and binarized (black versus white); total black pixels per field were counted, and average values were calculated for each sample. Three replicate samples and at least four fields per replicate were subjected to densitometric analysis. We performed the quantification of pixels per 100 cells and not per area in order to take into account the different cell morphology and confluence.

Cell-containing microfibers were fixed in glutaraldehyde 2.5% buffered solution and osmium tetroxide 2% buffered solution and dehydrated through an ethanol gradient; samples were araldite embedded (ACM Fluka Sigma-Aldrich Co., St. Louis, MO, USA) and the ultra-thin sections of a selected area were contrasted with uranyl acetate lead citrate and observed at transmission electron microscopy (TEM; ZEISS EM 910 electron microscope; Zeiss, Oberkochen, Germany). For toluidine staining, 5 µm sections from the same specimens were obtained with a glass blade. Sections were stained with toluidine blue, mounted in glycerol, and observed using a Leitz microscope.

Cryopreservation of Af, AGf, and AUBMf embedded chondrocytes was performed by adapting a previously published protocol (Pravdyuk et al., 2013). Microfibers embedded cells were directly freezed in standard culture medium with 10% FCS and supplemented with 10% of DMSO. The samples were cryopreserved at −20°C for 30 min, maintained at −80°C for 24 h, and then immersed in liquid nitrogen, where they were kept until the thawing day. Cryovials within microfibers were thawed in a warm water bath (37°C) and, when ice was totally melted, microfibers were washed twice with culture medium and subsequently cultured in standard conditions prior to performing morphological, viability, and immunocytochemical analysis.

All cell-related experiments were repeated with chondrocytes derived from five different donors and performed in triplicate for each donor. Data are presented as means ± SEM. The normal distribution of data was verified using the Kolmogorov–Smirnov test. In case of single comparison, statistical significance was determined by paired Student’s t-test for normally distributed data and Wilcoxon matched-pairs signed-ranks test for non-normally distributed data. In case of multiple comparisons, statistical significance was analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test if the values followed a normal distribution, or by Kruskal–Wallis analysis (non-parametric one-way ANOVA) and Dunn’s post hoc test if the values were not normally distributed. For all statistical analyses, differences were considered statistically significant for p-values ≤0.05.

Alginate (Af), alginate-gelatin (AGf), and alginate-UBM (AUBMf) microfibers were produced by a microfluidic procedure by a snake micromixing chip, consisting in two inlets, a mixing element, and an outlet tube with an internal diameter of 700 µm (Figure 1A). The dimensional analysis of photomicrographs of microfibers produced at different flow rates (from 0.5 to 1.5 mL/min) demonstrates that the microfluidic procedure allows a good control on microfibers’ morphology and dimensions (Figures 1B,C). Microfibers present a smooth surface and a uniform morphology, with constant dimensions (in diameter); particularly, the pumping rate has an important effect of the microfiber dimensions. High pumping rates resulted in an increase in microfiber diameters; this behavior is indeed attributed to the well-known Barus effect (Malkin et al., 1976). Moreover, the analysis of the photomicrographs reported in Figures 1B,C demonstrates that the addition of gelatin or UBM does not affect the general microfiber morphology (i.e., in term of dimension and surface smoothness). Both gelatin and UBM caused only a moderate enlargement of the microfiber diameter. The produced microfiber presents good structural and morphological properties as proved by the Coomassie Blue Brilliant staining (Figure 2). Particularly, AGf are characterized by uniform staining, since gelatin is homogeneously present within the entire microfiber structure (i.e., being gelatin soluble in the aqueous sodium alginate); on the contrary, AUBMf display the presence of UBM particles evenly distributed within the microfiber matrix arrowed in Figure 2 (i.e., being UBM particles unsoluble in water). Therefore, the images reported in Figure 2 indicate that the snake micromixing chip allowed a homogeneous distribution of gelatin and ECM along the whole microfiber.

With respect to the concentrations of biomaterial used for the preparation of microfibers (sodium alginate, gelatin, and UBM), the selected amounts were chosen on the basis of a large number of previously published observations (Penolazzi et al., 2010, 2012; Mazzitelli et al., 2013; Angelozzi et al., 2015; Vecchiatini et al., 2015). For instance, alginate is typically used in the concentration range comprised between 0.5 and 2.5% (w/v) depending on the stiffness required by the specific application of the gel.

To obtain dedifferentiated cells, P0 chondrocytes from five different donors were expanded up to six passages (Figure 3). As expected (Caron et al., 2012), the cartilaginous phenotype was progressively lost over several passages in culture. In fact, compared to P0 freshly isolated chondrocytes, P6 expanded chondrocytes exhibited a substantial change in cell morphology, from rounded to flattened fibroblast-like shape as it is evident from hematoxylin staining. Immunohistochemical analysis showed that expansion dramatically decreased the expression of typical chondrogenic markers including collagen type II (Col2A1), aggrecan, Sox9, and, in contrast, increased the positivity for collagen type I (Col1A1). Dedifferentiation status was confirmed by a dramatic decrease of sulfate GAG content in ECM composition (as demonstrated by Alcian Blue staining).

P6 chondrocytes expanded as dedifferentiated chondrocytes were then embedded in composite microfibers, which were subjected to specific analyses at days 7 and 14, as described below.

Dedifferentiated chondrocytes were suspended, at 2 × 10⁶ cells/mL in (i) alginate 2%, (ii) alginate 2% plus gelatin 2.25%, or (iii) alginate 2% plus UBM 0.5%. The different cells/biomaterial suspensions were used to produce microfibers (Af, AGf, and AUBMf) by letting flow the cell suspension in a BaCl₂ gelling bath at a pumping rate of 1.5 mL/min, resulting in microfibers with diameters comprised between 650 and 750 µm. The obtained alginate-based microfibers were maintained in culture medium without chondrogenic inducers up to 14 days, and the embedded cells were analyzed for viability at different time of culture. As shown in the lower part of Figure 3, after embedding in microfibres, cell viability was not compromised as confirmed by double staining with Calcein-AM/PI.

The properties of the embedded cells were further investigated in term of cell morphology, and proteoglycan deposition, by toluidine blue metachromasia of matrix. As shown in Figure 4, the embedded cells revert to a spherical shape in just 7 days. Interestingly, during the entire period of culture, up to 14 days, the embedded cells are mainly present as single cells, well dispersed in the alginate, showing an aspect resembling the environment of mature chondrocytes in cartilage. In addition, the presence of metachromatic areas with secretory vesicles in a well-defined pericellular space at days 7 and 14 was clearly detectable, indicating the effective release and deposition of cartilage-like ECM. The Af, AGf, and AUBMf composite microfibers represent therefore a microenvironment supporting the maturation of chondrocyte phenotype also in the absence of chondrogenic inducers.

Transmission electronic microscope ultrastructural analysis of embedded cells at day 14 revealed a high cellular secretory activity. As shown in Figure 5, the presence of secretory vesicles containing ECM dense materials, and collagen fibers with their typical banding pattern is clearly appreciable. The released materials accumulated and assembled in a sparse matrix in the surrounding lacuna, resembling the biological microenvironment of chondrocytes. These preliminary evidences confirmed the deposition of a cartilage-like ECM in this area, as hypothesized after the toluidine blue staining (see Figure 4). Interestingly, AUBMf showed the presence of cells able to interact with each other and with UBM flakes (characterized by typical collagen fibers) located in the closed areas.

The chondrogenic properties of the embedded cells were then investigated with two different approaches (Figures 6 and 7). In a first step, embedded cells were harvested from microfibers at days 7 and 14 of culture and subjected to evaluation of chondrogenic markers. RT-qPCR revealed that the main cartilage-specific ECM component, Col2A1, and the major proteoglycan in cartilage, aggrecan, were highly expressed in cells embedded in Af, AGf, and AUBMf compared to chondrocytes cultured in standard micromass (MM) and P6 dedifferentiated chondrocytes (Figure 6A). Micromass culture system has been chosen instead of pellet culture since it may be maintained in culture medium also without adding TGFβ supplementation. The remarkable increase of Col2A1 expression and decrease of Col1A1 was particularly appreciable calculating the ratio between Col2A1 and Col1A1 (differential index) which significantly increased from 20 to 120 times in all embedded cells compared to MM cultured cells (Figure 6B). Moreover, gene expression analysis showed also the absence of expression of Col10A1, which encodes the collagen type 10 alpha 1 chain traditionally associated with chondrocyte hypertrophy, both at 7 and 14 days (Figure 6B). This suggested that microfiber environment is suitable to prevent undesired hypertrophic maturation that can widely affect the successful outcome of the graft transplantation. The reacquistiton of chondrogenic properties by the embedded cells was confirmed by GAG quantification performed as total GAG content normalized to DNA content (Figure 6B), and comparable GAG content was found in Af, AGf, and AUBMf embedded cells.

It is important to underline that with these experiments we also demonstrated that the small amount of TGFβ in the FCS containing medium is not sufficient to support chondrogenesis in standard conditions of MM that requires exogenous chondrogenic inducers. Conversely, microenvironments created by microfibers support redifferentiation capacity of expanded chondrocytes without the need of adding chondrogenic inducers.

In a second step, embedded cells were recovered at day 14 from the microfibers, reseeded, and grown up to 7 days as monolayered culture in standard medium without adding chondrogenic inducers. These experiments were aimed both at validating the previous RT-qPCR data by immunocytochemical analysis and at further demonstrating the intrinsic potency of microfibers in maintaining chondrogenic activity of the cells once released from the confined 3D microenvironment of the scaffolds.

Interestingly, even if plated in unfavorable conditions, the cells maintained a round morphology, a low adhesion ability, and generated microaggregates, demonstrating a behavior comparable to freshly isolated chondrocytes. As shown in Figure 7, the cells were intensively stained by Alcian blue for sulfated GAGs and immunostained for Col2A1. The presence of UBM was particularly effective in promoting the maintenance of chondrogenic phenotype as evidenced by a larger number of Col2A1 positive spontaneous microaggregates that persisted over 7 days.

To explore the possibility to set up a bank of “microfibrous scaffolds embedded chondrocytes” for further in vitro and in vivo manipulations, we performed a preliminary assessment of the properties of thawed Af, AGf, and AUBMf embedded cells after freezing procedure. The redifferentiated chondrocytes were frozen directly within microfibers in complete culture medium supplemented with 10% DMSO, stored at −196°C in liquid nitrogen, thawed, and then assessed for cellular viability and chondrogenic properties. Similar to the freshly Af, AGf, and AUBMf embedded cells, thawed samples maintained highly viable cells with a round shaped morphology (Figure 8). When recovered from microfibers and grown in monolayer in standard medium without chondrogenic inducers for 24 h (the conditions are the same described in Figure 7), thawed samples preserved GAG production and Col2A1 expression as shown by Alcian Blue staining and immunocytochemistry (Figure 8).

Therefore, the alginate-based microfibers appear resistant to freezing and allow us to recover highly viable and functional cells also after thawing.