Response of human chondrocytes and mesenchymal stromal cells to a decellularized human dermis

Decellularized human dermis (HDM_derm) was taken from the back of four multi-organ and/or multi-tissue donors by following strictly Directive 2004/23/EC and Italian Transplant Center Guidelines on harvesting, processing and distributing tissues for transplantation.

In sterile conditions, using an electric dermatome, the epidermis was separated from the dermis trunk area and dermis samples of about 10 cm² were dissected. The thickness of the dermal samples was about 0.8 mm. The dermis grafts were rinsed with 0.9% NaCl solution and stored in this solution for transport (2-4°C) to the treatment station (< 12 hrs) where they were submitted aseptically to a combined treatment of decellularization. The tissue was treated with 2.5% trypsin (GIBCO, catalogue n. 15090) and placed in an incubator overnight (5% CO₂ at 37°C), washed twice with sterile 0.9% NaCl to remove trypsin remnants, then dipped in RPMI medium (containing 10000 IU/ml penicillin, 10 mg/ml streptomycin, 25 μg/ml amphotericin B) for 15 minutes and sealed inside cryofreezing bags (Hemofreeze bag – DF 700–3 by Fresenius Kabi) without adding cryoprotectors. Finally, the dermis was submitted to irradiation with γ rays (100 Gy) and stored in nitrogen vapour at -180/-190°C. This method provides a decellularized ECM scaffold, as shown by histology (H&E, DAPI stain), Transmission Electron Microscopy, cell viability test and DNA amount (Picogreen DNA Assay) [6].

Normal human articular chondrocytes (NHAC-kn, Clonetics™ Cell System, BioWhittaker Italia, Caravaggio, Italy), derived from a single-donor knee articular cartilage, and human mesenchymal stromal cells (Poietics™ hMSC, Lonza Walkersville Inc., Walkersville, USA) harvested and cultured from bone marrow of a single donor, were used for the experiment. NHAC-kn and hMSC cells were expanded using the Chondrocyte Growth Medium (CGM, containing fetal bovine serum 5%, gentamicin sulphate-amphotericin B 0.1%, b-Fibroblast Growth Factor 0.5%, R3-Insulin-like Growth Factor-1 0.2%, insulin 0.2%, transferrin 0.1%) and the Mesenchymal Stem Cell Growth Medium (MSCGM, containing 10% FCS, 30 μg/ml Gentamicin and 15 ng/ml Amphotericin, Lonza Walkersville Inc., Walkersville, USA). Four different batches of HDM_derm were aseptically cut into 10 × 10 mm² pieces; NHAC-kn, at passage 1, or hMSC were seeded onto forty-eight specimens at a density of 1 × 10⁵ cells. The same concentration of NAHC-kn and hMSC cells was seeded in empty polystyrene wells as control (CTR). After 24 h incubation, of growth media were changed with Chondrocyte Differentiation Medium (CDM, containing fetal bovine serum 5%, gentamycin sulphate-amphotericin B 0.1%, Transforming Growth Factor β-3 0.5%, R3- Insulin-like Growth Factor-1 0.2%, insulin 0.2%, transferrin 0.2%, and ascorbic acid 2.5%) in NHAC-kn cultures and with Complete Chondrocyte Induction Medium (Lonza Walkersville Inc., Walkersville, USA) in hMSC cultures. After 24 h of incubation, the medium was collected and non-adherent cells were counted. Cell adhesion was calculated as the difference between seeded cells and non-adherent cells, present in the medium and attached to the 24-well plate bottom, expressed as a percentage of the number of the seeded cells. At 7 and 14 days, the supernatant was collected and the following tests were performed: procollagen type II C-propeptide (CPII ELISA, IBEX Technologies Inc., Montreal, Quebec, Canada) and aggrecan (Human Aggrecan EASIA™ ELISA kit, BioSource Europe SA, Nivelles, Belgium) for phenotype expression, the multifunctional peptide transforming growth factor- β1 (TGF-β1, quantikine human TGF-β1 immunoassay, R&D Systems, MN, USA) for anabolic growth factor synthetic activity, interleukin1β (IL-1β, quantikine human IL-1β immunoassay, R&D Systems, MN, USA) and matrix metalloprotease 3 (MMP3, quantikine human MMP3 immunoassay, R&D Systems, MN, USA) for inflammatory and catabolic response assessment, respectively. The measured protein concentrations were normalized by Total Protein concentration (Total Protein Kit, Micro Lowry method, Petterson’s Modification, SIGMA®, Missouri, USA). The cell proliferation reagent WST-1 (Roche Diagnostic GmbH, Penzberg, Germany) test was performed and quantified spectrophotometrically at 450 nm with the reference wavelength at 625 nm. Results were expressed as % Vitality calculated as follows:

where ODs was the optical density of the sample and OD CTR was the optical density of control wells.

After 14 days of cell culture, the seeded membranes were studied to evaluate chondrocyte and mesenchymal stromal cell colonization inside HDM_derm by histology and scanning electron microscopy (SEM). For histology, specimens were fixed in 10% buffered formalin solution in PBS (pH 7.4), embedded in paraffin and sections were cut to 6-μm thickness and stained with Haematoxilin & Eosin, Safranin O and Alcian Blue. Images were grabbed by using an Olympus BX51 microscope equipped with XC30 Olympus camera. For SEM, specimens were fixed in 2.5% v/v glutaraldehyde (pH 7.4 in PBS 0.01M, 1h) and dehydrated in graded ethanol series. After a passage in hexamethyldisilazane, the samples were air dried, then sputter-coated with gold before being examined by a Philips XL-20 SEM.

Statistical evaluation of data was performed using the SPSS/PC+ Statistics TM 12.1 (SPSS Inc., Chicago, IL USA) software package. Data are reported as Mean ± SD at a significance level of p < 0.05. After checking normal distribution and homogeneity of variance, two-way ANOVA (group and experimental time) followed by Bonferroni’s t test were used to compare results. Student’s t test was used to compare cell vitality index results.

Tables 1 and 2 show the results of NHAC-kn and hMSC cells seeded on the HDM_derm membrane at 7 and 14 days. The phenotypic expression given by the synthesis of CPII did not differ within the two cell types. The synthesis of CPII on HDM_derm increased between experimental times for both cell types (NHAC-kn, p < 0.05; hMSC, p < 0.05). At both experimental times, HDM_derm induced NHAC-kn to produce almost double the amount of CPII than did CTR (p < 0.05). The synthetic activity, expressed by the measured concentration of aggrecan, increased over time in both cells (p < 0.0005). NHAC-kn (14 days) and hMSC (7 and 14 days) seeded onto HDM_derm produced significantly higher amounts of aggrecan than those seeded on CTR (p < 0.0005). Regarding the synthesis and release of TGF-β1, significant decreases were found for NAHC-kn (p < 0.05) and hMSC (p < 0.0005) in the CTR group between experimental times. For hMSC, significantly higher TGF-β1 values were found when cultured on HDM_derm than those found on CTR at both 7 and 14 days (p < 0.0005). Concerning inflammatory responses, no statistically difference in the release of IL-1β was observed for both cells and experimental times. At 7 days NHAC-kn seeded onto HDM_derm produced significantly lower values of MMP-3 compared with CTR (p < 0.005). Furthermore, the MMP-3 released by NHAC-kn in CTR was observed to decrease significantly over the culture time (p < 0.005).

Significant phenotypic marker expression data obtained from NHAC-kn and hMSC cultures seeded on HDM_derm were normalized to those of their controls (Table 3). When comparing normalized data, NHAC-kn showed a significant increase in CPII synthesis with respect to that showed by hMSC cells, approximately 2.2 fold, at both experimental times (p < 0.005), whereas hMSC cells induced a significant increase in aggrecan at 7 days (1.6 fold, p < 0.005) and TGF-β1 at both experimental times (approximately 1.5 fold at 7 days and 2.3 fold at 14 days) compared with that induced by NHAC-kn cells (p < 0.005).

The main histo-architecture of the acellular extracellular matrix was retained. After 14 days in static culture, both cells grew prevalently on the HDM_derm surface. NHAC showed rounded features forming aggregates along the HDM_derm surface and penetrating in the deeper layers of the decellularized membrane (Figure 1a). hMSC exhibited a fibroblast-like morphology with elongated nuclei covering the membranes, whereas occasional cells were observed to infiltrate more deeply (Figure 1b). Safranin O (Figures 1c,d) and Alcian blue (Figures 1e,f) stainings showed proteoglycan synthesis, only at intracellular level, and the presence of GAGs deposited on the decellularized matrix just in the proximity of grown cells, respectively, without differences between NHAC-kn and hMSC.

SEM analysis performed 14 days after seeding on HDM showed different patterns for NHAC-kn and hMSC, respectively. A homogeneous and multilayered surface-covering colonization with possibly living cells was found for NHAC-kn on HDM_derm: they had a polygonal-rounded morphology or fusiform shape. Most of the cells were connected to each other by forming cell aggregations and were tightly interwoven within the collagen network. Conversely, hMSCs partially covered HDM_derm by cell aggregates associated with a clear-cut collagen meshwork (Figure 1g,h).