Chondrogenic differentiation induced by extracellular vesicles bound to a nanofibrous substrate

Isolation and expansion of hACs

Human cartilage samples were collected under Informed Consent from patients undergoing knee arthroplasty (female; aged 69 years) in the Hospital Center of Alto Ave, Guimarães, Portugal, in accordance with the established Protocol (67/CA). After the surgeries, a small part of the non-compromised knee cartilage was sectioned and preserved in phosphate-buffered saline (PBS) at 4 °C until the subsequent isolation procedure of the hACs⁵⁶. Briefly, collected cartilage samples were dissected in small full-depth pieces, washed, and digested with 0.25% w/v trypsin solution (Sigma-Aldrich) for 30 min at 37 °C under agitation. Then the solution was removed, and the cartilage was washed and incubated overnight at 37 °C with a 2 mg mL⁻¹ collagenase type II solution (Sigma-Aldrich). In the following day, the cells were washed, counted, and plated at a density of 2 × 10⁶ cells. Cells were expanded in expansion medium [EM; Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) containing 10 mM Hepes buffer (Sigma-Aldrich), L-alanyl-L-glutamine (Sigma-Aldrich), non-essential amino acids (Sigma-Aldrich), 1% antibiotic–antimycotic solution, 10% fetal bovine serum (FBS), and 10 ng mL⁻¹ basic fibroblast growth factor (bFGF; PeproTech)] at 37 °C in a humidified atmosphere of 5% CO₂.

Isolation and expansion of hBM-MSCs

Human bone marrow aspirates were collected, after obtaining informed consent from patient undergoing knee arthroplasty (female; aged 55), under the cooperation agreement established between the 3Bs Research Group, University of Minho and the Hospital Center of Alto Ave, Guimarães, Portugal. hBM-MSCs were isolated and characterized using our established standard protocols⁵⁷. Cells were expanded in BM [MEM alpha medium (α-MEM; Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Life Technologies) and 1% antibiotic/antimycotic solution (final concentration of penicillin 100 units mL⁻¹ and streptomycin 100 mg mL⁻¹; Life Technologies)] at 37 °C in a humidified atmosphere of 5% CO₂.

Conditioned media

Conditioned media harvested from the hACs and hBM-MSCs in culture were used as a source of EVs, according to the schematic illustration of the experimental design (Fig. 7). First, the hACs and hBM-MSCs were expanded at passage 3 and a cell suspension containing of 3 × 10³ cells cm⁻² were subcultured in T150 flasks and cultured in EM and BM, respectively, at 37 °C in a humidified atmosphere of 5% CO₂. The conditioned media were harvested when the cells reach the confluence (in 7 days), pooled, filtered (pore size 0.22 μm) to remove cell debris, and stored at −80 °C until further use. A cell suspension containing 2 × 10⁵ cells/15 mL centrifuge tubes of hACs or hBM-MSCs were also prepared to assure obtaining conditioned medium of chondrogenic lineage commitment. In order to form a spherical aggregate or pellet cultures, the cell aliquots were centrifuged at 600 × g for 5 min and incubated at 37 °C in a humidified atmosphere of 5% CO₂ for 24 h incubation. The hACs and hBM-MSCs were cultures under DM [EM—instead of adding bFGF, 1 mg mL⁻¹ L-ascorbic acid (Sigma-Aldrich) and 1 mg mL⁻¹ insulin (Sigma-Aldrich) were added] or standard chondrogenic DM [CM; BM supplemented with Insulin-Transferrin-Selenium-G Supplement (ITS; Invitrogen), 1 mM dexamethasone (Sigma-Aldrich), 0.1 M sodium pyruvate (Invitrogen), 17 mM ascorbic acid-2-phosphate (Sigma-Aldrich), 35 mM L-proline (Sigma-Aldrich), and 10 ng mL⁻¹ transforming growth factor-β3 (PeproTech)], respectively. Those conditioned media (DM; CM) were harvested at 21, 24, and 28 days of culture, pooled, filtered (pore size 0.22 μm) to remove cell debris, and stored at −80 °C until further use.

The amount of EVs presented on each conditioned medium was quantified by the Exosome ELISA Complete Kit (CD63) (System Biosciences) after EV isolation using a polymeric precipitation solution (ExoQuick-TC; System Biosciences, BioCat GmbH). Those assays were performed according to the manufacturer’s instructions.

The size of the isolated EVs was determined by dynamic light scattering at an angle of 173° and at a wavelength of 633 nm, and the zeta-potential was determined by laser Doppler electrophoresis using a Zetasizer Nano ZS instrument (Malvern Instruments).

Preparation of activated and functionalized PCL NFMs

The production of electrospun PCL NFMs was performed as described in detail elsewhere⁸. In brief, a polymeric solution of 15% (w/v) PCL (Mn 70,000–90,000 by GPC, Sigma-Aldrich) in chloroform (Sigma-Aldrich) and N,N-dimethylformamide (7: 3 volume ratio; Sigma-Aldrich) was electrospun at 12 kV, using a needle-to-ground collector distance of 20 cm, and a flow rate of 1.0 mL h⁻¹. The electrospun NFM is composed of nanofibers with diameters in the micrometer range, from 0.4 to 1.4 μm, with an average pore size of 7.267 ± 3.148 μm and a thickness range from 40 to 60 μm⁵⁸.

Samples of electrospun PCL NFM (1 cm²) were activated in an ultraviolet−ozone system (ProCleaner 220, Bioforce Nanoscience) by exposing both sides for 2 min each. Incubation in 1 M 1,6-hexanediamine solution (Sigma-Aldrich) for 1 h at 37 °C was performed, in order to graft amine groups (-NH₂) at the NFM surface.

Engineered EV immobilization systems

Since EVs, namely, exosomes, typically express the CD63 surface marker, the human anti-CD63 antibody (E-12; Santa Cruz Biotechnology, Inc.) was immobilized at the surface of NFMs by a covalent bond mediated by a coupling agent, namely, 1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide/hydroxysuccinimide mixture (10 mM EDC + 40 mM NHS; Sigma-Aldrich, S.L.). The antibody solution (1% (v/v)) was mixed for 15 min at room temperature (RT), for the antibody activation, and incubated 2 h at RT on the activated and functionalized nanofibrous substrate.

The maximum immobilization capacity of the antibody over the nanofibrous substrate was determined by using a wide range of concentrations (from 0 to 8 μg mL⁻¹). After the anti-CD63 antibody immobilization, a blocking step was performed by a 3% bovine serum albumin (BSA; Sigma) for 1 h at RT, followed by the secondary antibody (1:200 in PBS) incubation (1 h at RT). Alexa Fluor^® 488 rabbit anti-mouse (~495/517 nm; Life Technologies) was used as secondary antibody against the anti-CD63 antibody. The unbound secondary antibody fluorescence was measured in a microplate reader (Synergy HT, Bio-TEK), as an indirect method to determine the primary antibody immobilization efficiency. In order to evaluate nonspecific immobilization, the activated and functionalized NFM without primary antibody was used as a negative control. The samples were further analyzed by fluorescence microscopy (Axio Observer; Zeiss) to detect the distribution of the anti-CD63 antibody at the surface of the nanofibrous substrate.

Biofunctionalization of NFMs with EVs was achieved by using an antibody–antigen strategy, as described in detail elsewhere⁵⁹. The nanofibrous substrate with immobilized anti-CD63 antibody, at the antibody concentration previously optimized, was incubated with the different conditioned medium previously harvested (i.e., AC^EM, AC^DM, MSC^BM, MSC^CM) for 2 h at RT. The unbound EV solutions were collected and quantified by enzyme-linked immunosorbent assay (ELISA; Exosome ELISA Complete Kit (CD63)), in order to define the binding capacity of the engineered EV immobilization system. The negative control samples (i.e., non-biofunctionalized NFM) were performed by carrying out all the biofunctionalization steps (including the incubation step with different conditioned media) but substituting the anti-CD63 antibody solution by the PBS solution.

The morphology of EVs bound at the surface of biofunctional electrospun PCL NFM (i.e., NFM/EV-AC^EM, NFM/EV-AC^DM, NFM/EV-MSC^BM, NFM/EV-MSC^CM) was analyzed by SEM (AURIGA Compact, Zeiss, Germany). By EDS (INCAx-Act, PentaFET Precision, Oxford Instruments), an elemental analysis of the EV immobilization systems was performed to further confirm the presence of the EVs at the surface of the NFMs.

The distribution of EVs bound to the NFM biofunctionalized with anti-CD63 antibody was performed by immunofluorescence staining. First, a blocking step (3% BSA for 30 min) was performed. In between each step, the samples were rinsed three times in PBS buffer. For EVs’ surface marker staining, samples were incubated with the primary antibodies CD63 (E-12; 1:500; Santa Cruz Biotechnology, Inc.), CD81 (1.3.3.22; 1:500; Santa Cruz Biotechnology, Inc.), and CD9 (C-4; 1:500; Santa Cruz Biotechnology, Inc.) overnight and then with the corresponding secondary antibody [Alexa Fluor^® 488 rabbit anti-mouse (~495/517 nm; Life Technologies)] for 1 h at RT. The non-biofunctionalized NFM was used as a negative control to evaluate nonspecific immunodetection of EVs’ surface markers in the biofunctionalized electrospun NFMs. The samples were further analyzed by fluorescence microscopy (Axio Observer; Zeiss).

Proteomic analysis of the bound EVs

The total protein content of EVs bound to the surface of biofunctional NFM were prepared using RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma) for 1 h at 4 °C. The samples were subjected to ultrasonic cracking, centrifuged for 15 min at 14,000 rpm at 4 °C, and the supernatants were collected.

Protein extracts of 15 μg were solubilized with 100 mM Tris pH 8.5, 1% sodium deoxycholate, 10 mM tris(2-carboxyethyl)phosphine and 40 mM chloroacetamide for 10 min at 95 °C at 1000 rpm (Thermomixer, Eppendorf). Each sample was processed for proteomic analysis following the solid-phase-enhanced sample-preparation (SP3) protocol as described in detail elsewhere⁶⁰. Enzymatic digestion was performed with Trypsin/LysC overnight at 37 °C at 1000 rpm.

Protein identification and quantitation was performed by nanoLC-MS/MS as described in detail elsewhere⁶¹. The raw data was processed using the Proteome Discoverer 2.5.0.400 software (Thermo Scientific) and searched against the UniProt database for the Homo sapiens Proteome (2020_05 with 75069 entries), the NIST human spectral library, and Bos taurus Proteome (2020_05 with 37512 entries). A common protein contaminant list from MaxQuant was also considered in the analysis. The MSPepSearch and Sequest HT search engines were used to identify tryptic peptides. The ion mass tolerance was 10 ppm for precursor ions and 0.02 Da for fragmented ions in both softwares. Maximum allowed missing cleavage sites was set to two. Cysteine carbamidomethylation was defined as constant modification. Methionine oxidation, deamidation of glutamine and asparagine, peptide terminus glutamine to pyroglutamate, and protein N-terminus acetylation, Met-loss, and Met-loss+acetyl were defined as variable modifications. Peptide confidence was set to high. The processing node Percolator was enabled with the following settings: maximum delta Cn 0.05; decoy database search target false discovery rate 1%, validation based on q-value. Protein label-free quantitation was performed with the Minora feature detector node at the processing step. Precursor ions quantification was performed at the processing step with the following parameters: peptides to use all peptides, precursor abundance based on intensity, and normalization based on total peptide amount. Protein ratio was based on protein abundance and an analysis of variance (Individual Proteins) hypothesis test was performed.

Bioinformatic analysis

The identified proteins were analyzed using the Venny tool (v2.1.0, https://bioinfogp.cnb.csic.es/tools/venny/). The sequences of the identified proteins were mapped according to their GO to determine their biological and functional properties, using InterProScan (v.5.14-53.0, http://www.ebi.ac.uk/interpro/). Proteins were grouped with regard to the biological process, cellular component, and molecular function using the categories of the Panther Biological process (v.15.0, http://www.pantherdb.org/). The GO analysis and heat map were obtained using the GraphPad PRISM v. 8.0. Clusters heat map were assembled by GO analysis using an Euclidean distance function.

Cell seeding and culture conditions

The effectiveness of the developed EV immobilization systems, as chondrogenic lineage inducible systems, was assessed using hBM-MSCs (Table 1). Confluent hBM-MSCs at passage 4 were harvested for seeding on top of the EV immobilization systems (NFM/EV-AC^EM, NFM/EV-AC^DM, NFM/EV-MSC^BM, NFM/EV-MSC^CM) at a density of 2 × 10⁵ cells per sample. These constructs were cultured under BM, without further medium supplementation. The experimental control conditions comprise hBM-MSCs cultured on top of the non-biofunctionalized NFMs under BM (NFM_Ctrl−) and using the standard chondrogenic DM (NFM_Ctrl+). hACs were also seeded on top of the non-biofunctionalized NFMs at a density of 2 × 10⁵ cells per sample and cultured under EM (NFM_EM) or DM (NFM_DM) (Supporting Information section—Supplementary Figs. 4 and 5). The constructs were retrieved for further analysis at predefined culturing times, namely, 0, 7, 14, 21, and 28 days. All experiments were performed in triplicate and repeated at least three times (n = 3), independently.

Cellular biochemistry analysis

The metabolism was evaluated by the MTS assay (CellTiter 96 AQ_ueous One Solution, Promega), the cell proliferation by DNA quantification (Quant-iTPicoGreen dsDNA assay, Invitrogen, Alfagene), and the protein synthesis by the Micro BCA assay (Micro BCA^TM Protein Assay Kit, Thermo Fisher Scientific), according to the manufacturers’ instructions. GAG quantification was performed according to our established standard colorimetric assay⁸.

Scanning electron microscopy

The constructs were collected after 28 days of culture and fixed with 2.5% glutaraldehyde. By increasing the alcohol concentrations, samples were dehydrated, followed by sputter coating with Au/Pd. A scanning electron microscope (JSM-6010 LV, JEOL, Japan) was used to observe the distribution and morphology of the cells at ×3000 magnification.

Gene expression analysis

At each time point, the constructs were washed, immersed in Tri reagent^® (Life Science, VWR), and kept at −80 °C. Total RNA was isolated and reverse transcribed into cDNA (qScript cDNA synthesis kit, Quanta Biosciences), followed by quantitative polymerase chain reaction (qPCR; PerfeCta^TM SYBR^® Green system; Quanta Biosciences), according to the manufacturer’s instructions. The qPCR reactions were carried out in a Mastercycler^® ep Gradient S realplex^® thermocycler (Eppendorf; Hamburg) for the target genes described in Table 2. The transcript expression data were normalized against the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase and the quantification was performed according to the Livak method (2^−ΔΔCT method). For hBM-MSC samples, the BM condition (NFM_Ctrl-) was used as calibrator, while the EM condition (NFM_EM) was used as calibrator of the samples of hACs.

Histological analysis

Constructs were collected after 28 days of culture, fixed in a 4% paraformaldehyde solution, and kept at 4 °C until further used. The staining procedures were performed on top of the samples. Alcian blue staining, were performed as described elsewhere⁶². For immunohistochemistry, the samples were permeabilized using 0.1% Triton X-100 in PBS for 15 min, incubated with 3% BSA, and incubated with a defined primary antibody (collagens type I (COL1A1, clone C-18; Santa Cruz Biotechnology) and type II (mouse anti-human type II collagen monoclonal antibody; Millipore); actin (mouse anti-alpha smooth muscle Actin antibody; Abcam)) overnight at 4 °C. The samples were then washed with PBS and stained with the corresponding secondary antibodies (Life Technologies). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (1 mg mL⁻¹ in PBS for 15 min). Fluorescence images from stained constructs were obtained using a confocal laser scanning microscope (Leica TCS SP8).

Statistical analysis

Statistical analysis was performed using the SPSS statistic software (release 24.0.0.0 for Mac). First, Shapiro–Wilk test was used to ascertain the data normality and Levene test for the homogeneity of variances. Observing this, the normality and variance homogeneity were rejected; non-parametric tests were used (Kruskal–Wallis test followed by Tukey’s Honest Significant Difference test). The confidence interval used was 99% and p ≤ 0.01 were regarded as statistically significant.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of Minho Life Sciences Ethics Committee (SECVS 136/2015), the Hospital Center of Alto Ave, Guimarães, Portugal (67/CA), and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.