Rheumatoid arthritis (RA) is an autoimmune disease that affects primarily the synovial cavity tissues on the small diarthrodial joints of the hands and feet [1
], leading to persistent synovial inflammation [2
] and progressive erosion of the articular structures [3
]. The triggers for the disease susceptibility and the pathological cascade of events encompass environmental, genetic and stochastic factors [3
]. For example, RA has special incidence on females [5
], which is related with genetic [5
] and hormonal factors [5
], but also presents a heterogeneous geographic distribution, being less common in developing countries [11
], which confirms the involvement of environmental and socio-cultural factors [12
The disease pathophysiology involves several interconnected mechanisms. Specific antibodies for immunoglobulin G (IgG) mediate the autoimmune process, as well as the imbalanced expression of the pro-inflammatory cytokine’s profile and its functionality [1
]. Consequently, this leads to inflammatory processes, autoimmunity enhancement, long-term inflammatory synovitis and joint damage [13
]. Additionally, locally expressed degradative enzymes digest the cartilaginous matrix and destroy the articular surfaces [1
]. Infiltration of B cells, CD4+ T cells and macrophages into the synovium, which in normal conditions is relatively acellular [1
], leads to soft tissue oedema and stiffness [2
]. Moreover, other inflammatory cells such as neutrophils, natural killer cells and mast cells play key roles in the disease’s progression [13
From the autoimmune disease (AD) subsets that entail the multiple inflammatory cascades, one of the most significant players is the TNF-α, particularly the over-expression of the TNF-α [11
]. TNF-α, a 233 amino acid protein [18
], is a key signaling cytokine in the immune system mainly produced by monocytes, macrophages [19
], and B and T cells [2
]. TNF-α stimulates the production of other inflammatory mediators, namely IL’s, as well as the recruitment of immune and inflammatory cells into the joint [20
]. As a regulatory cytokine that manages communication between immune cells and controls many of their functions when deregulated, TNF-α plays a key role in the pathogenesis of chronic inflammatory diseases, such as RA [22
]. TNF deregulation in RA is linked to TNF-α converting enzyme (TACE or ADAM17), a metalloproteinase that cleaves trans-membrane TNF, releasing the soluble segment [18
]; and to TNF receptors (mostly TNFr-I) [18
], which are likely to be related with proinflammatory, cytotoxic and apoptotic responses [2
The increasing knowledge on RA pathogenesis stimulated the development of different therapeutic modalities aiming to avoid joint destruction, minimize the symptomatic profile, and enhance physical function [27
]. These options comprise analgesics, symptomatic management and inflammatory drugs [11
]. Analgesics enclose the non-steroidal anti-inflammatory drugs (NSAIDs) that are commonly used to treat RA. The inflammatory suppressive drugs include glucocorticoids and disease-modifying anti-rheumatic drugs (DMARDs) both non-biologic and biologic. Due to the disease heterogeneity, therapeutic strategies must be tailored to the individual patient in order to achieve a low level of disease activity within a limited period of time [29
]. Thus, the combination of two treatment modalities has been proposed to achieve such a purpose [30
Considering the significant involvement of TNF-α in RA, as well as the rising evidences that this cytokine heads the pro-inflammatory cytokine cascade, it becomes a significant therapeutic target [31
]. Specifically, these evidences led to the development of TNF inhibitors for the treatment of ADs [33
], as they became the first class of biologic agents to be used in RA treatment [38
]. A specific high affinity monoclonal antibody is used to recognize and neutralize selectively its antigen, i.e., TNF-α [18
]. Five different types of TNF-α inhibitors are currently licensed for human clinical use in RA treatment, namely Infliximab, Entarnecept, Adalimumab, Certalizumab Pegol and Golimumab. With the exception of Infliximab, which is administrated by intravenous (IV) infusion, all other medicines are administered subcutaneously [13
]. For long-term control of RA, a continuous treatment is required because of disease flares’ risk when the therapy is discontinued [1
]. Although these therapeutic modalities have been used for quite some time, they present many shortcomings such as lack of specificity, limited antibody half-life, high cost, response variability, or even lack of response to treatments [11
]. Due to the systemic character of these treatments, not only target tissues but also healthy tissues are exposed to a significant dose of drug, leading to adverse side effects [41
]. These effects are common to many tissues, organs and systems, such as cardiovascular, renal, dermatological, and neurological, or risk of severe infection [11
Furthermore, some attention has been given to drug carriers, in order to maintain effective concentration levels in plasma for extended periods [41
]. Examples of these systems are the encapsulation of Infliximab in polylactide-co-glycolide microspheres [42
] or porous silicon 3D structures [43
], as well as long-term release of Etanercept by polyelectrolyte complex formulated particles [44
]. Despite the promising preliminary results, these systems are still poorly developed.
Considering the shortcomings of these treatments, there is a need for innovative approaches that circumvent the aforementioned adverse side effects. Therefore, the main objective of this work was to develop an implantable system capable of capturing excessive TNF-alpha present in intra-articular cavities of an RA patient. For that, we immobilized a neutralizing TNF-α antibody at the surface of a polymeric substrate (i.e., electrospun nanofibers). This strategy takes advantage of specific interactions between the antibody and the antigen, where the antibody (TNF-α antibody) binds to the antigen (TNF-α) avoiding/limiting its harmful pro-inflammatory effects. After the system´s assembly and proved ability to capture TNF-α secreted by activated monocyte-derived macrophages, the cytotoxicity of the biofunctionalized system was tested with human articular chondrocytes. Indeed, the cytocompatibility and chondrogenic differentiation potential of bare electrospun nanofibers were previously reported by us [45
], corroborating their potential use in cartilage regeneration approaches.
2. Materials and Methods
Polycaprolactone (PCL; Mn = 70,000–90,000 determined by Gas Permeation Chromatography), chloroform, N,N-dimethylformamide (DMF), 1,6-hexamethylenediamine (HMD), Ellmans reagent (DTNB), 2-iminiothiolane (2IT), 2-(N-morpholino)-ethanesulfonic acid (MES hydrate), phosphate buffered saline (PBS), N-hydroxysuccinimide (NHS), and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and kept at room temperature (RT), with the exception of 2IT which was kept at 4 °C and EDC which was kept at −20 °C. 4-(dimethylamino) pyridine (DMAP) was purchased from Millipore (Darmstadt, Germany) and kept at RT. Anti-TNF-α antibody [B-C7] was purchased from Abcam (Cambridge, UK) and kept at 4 °C until further use. Alexa Fluor® 488 goat anti-mouse rabbit IgG (H + L) and TNF-α human ELISA kit were purchased from Life Technologies (Carlsbad, CA, USA) and kept at 4 °C until further use.
2.2. Production and Functionalization of Nanofiber Meshes
2.2.1. Production of Nanofiber Meshes
Nanofiber meshes (NFMs) were prepared as described elsewhere [48
]. Briefly, the NFMs were produced by electrospinning of a polymeric solution of 15% (w
) PCL dissolved in an organic solvent mixture of chloroform and dimethylformamide (7:3 ratio). This PCL solution was electrospun by applying a voltage of 12 kV, a needle tip-to-ground collector distance of 20 cm and a flow rate of 1 mL/h. After the complete processing of 1 mL of PCL solution, the NFMs were left to dry for 1 day at RT inside a chemical hood, in order to completely evaporate the solvents. All further tests described were performed in samples of 10-mm squares.
2.2.2. Surface Functionalization of Electrospun NFMs
The surface functionalization of electrospun NFMs was performed as described elsewhere [48
]. Briefly, the NFMs’ surface was activated by 4 min of UV-Ozone irradiation (UV-O-Cleaner®
, ProCleaner 220, Bioforce Nanoscience, Salt Lake City, UT, USA). Afterwards, the amine groups (–NH2
) were inserted by immersion of the irradiated NFM in a 1 M hexanediamine (HMD) solution during 1 hour at 37 °C.
2.2.3. Quantification of Amine Groups Present at the Functionalized NFMs
Quantification of amine groups present in each NFM was performed as described elsewhere [50
]. For that, SH groups were inserted at the surface of the aminolysis-treated NFMs through the reaction of the amine groups with a 20 mM 2IT solution (0.1 mM PBS at pH8) and with a 20 mM DMAP solution during 1 h at 37 °C. Ellman’s reagent method was used to quantify the amino groups. For that, samples were immersed in 0.1 mM DTNB solution (in PBS 0.1 mM at pH 7.27) and incubated during 1 h at 37 °C. The absorbance of supernatants was measured in triplicate at 412 nm in a quartz plate, using the DTNB solution as blank. For the calculation of the 2-nitro-5-thiobenzoate (NTB−2
) molar absorption coefficient, the value of 14,151 M−1
was used [50
2.3. Antibody Immobilization
The TNF-α antibody was immobilized at the surface of activated and functionalized electrospun NFMs. To determine the substrate’s maximum immobilization capacity, a wide range of primary antibody concentrations were considered (from 0 to 12 µg/mL). The TNF-α antibody was firstly activated by a 15-minute incubation with a solution of EDC/NHS (50 mM EDC and 200 mM NHS), dissolved in a 0.1 M 2-(N
-morpholino)-ethanesulfonic acid (MES) buffer with 0.9% (w
) NaCl, followed by pH adjustment to 4.7 [48
The functionalized NFMs were incubated with 200 μL of the activated TNF-α antibody overnight at 4 °C. Biofunctionalized NFMs were washed three times with PBS. A 3% bovine serum albumin (BSA) solution was used as a blocking step. To determine the degree of TNF-α antibody immobilization, biofunctionalized NFMs were incubated with an Alexa Fluor® 488 solution diluted in PBS (1:200 ratio). As a negative control, NFMs with no antibody immobilized (plotted as 0 µg/mL) were used. The fluorescence of the supernatant was determined in triplicate, using an excitation of 495/20 nm and an emission of 519/20 nm, and used to quantify the amount of antibody effectively immobilized.
2.4. Characterization Biofunctionalized NFMs
2.4.1. Scanning Electron Microscopy (SEM)
SEM was used to analyse the morphology of biofunctionalized NFMs. Briefly, the biofunctionalized NFMs were sputter-coated with a thin layer (9–12 nm) of gold/palladium (Cressington 208 HR) and then analysed by SEM (NanoSEM, Nova 200, FEI company, Hillsboro, OR, USA). Micrographs were recorded at 15 kV with magnifications ranging from 100 to 2000 times.
2.4.2. Fluorescence Microscopy
A fluorescence microscope (Axio Imager Z1m, Zeiss, Gottingen, Germany) was used to analyze the spatial distribution of the TNF-α antibody at the surface of electrospun NFMs. The TNF-α antibody was linked by a secondary antibody with green fluorescence (Alexa Fluor™ 488). Photographs were recorded at magnifications of 50, 200 and 400 times.
2.5. Capturing of TNF-α Present in Conditioned Medium of Macrophage Culture
A human monocytic cell line THP-1 was maintained in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 µg/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES, and 10% fetal bovine serum (complete RPMI, cRPMI) (Life Technologies, Carlsbad, CA, USA). For the induction of cell differentiation, cells (106 per mL) were seeded in cRPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) for 24 h. After incubation, non-attached cells were removed by aspiration, and the adherent cells were washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype before stimulation, cells were incubated for an additional 48 h in cRPMI without PMA. For stimulation and retrieval of conditioned media, cells were further incubated for 4 h with 100 ng/mL of lipopolysaccharide (LPS) in fresh cRPMI and the supernatants were collected and stored at −80 °C. Production of TNF-α was assessed in the conditioned media by commercial ELISA (Life Technologies, Carlsbad, CA, USA). Four systems were tested at least in quadruplicates, in two independent assays: (1) the biofunctionalized NFM (with immobilized TNF-α antibody); (2) a positive control with the soluble TNF-α antibody; (3) a negative control with the UV-O activated NFM; and (4) the conditioned cell culture medium alone. Each system was placed in sterile Eppendorf tubes and 2 mL of monocyte-derived macrophage conditioned medium was added individually, and kept at 37 °C in agitation in an orbital shaker at 120 rpm. Initially, a 3 days assay was performed with a 1 ng/mL TNF-α concentration from monocyte-derived macrophage conditioned medium. A specimen was collected at the following time points (2, 4, 6, 8, 10, 12, 24, 32, 48, and 72 h of incubation) without conditioned medium replacement. Afterwards, a 15-day assay was conducted, where a sample was collected daily and recharged with the same volume of recovered cell culture medium each day, which had a TNF-α concentration of 500 pg/mL. This concentration was chosen due to being closer to TNF-α levels in active RA patients (76.1 ± 103.2 pg/mL) [19
], considering a margin of error affected by the TNF-α degradation.
Enzyme-Linked Immunosorbent Assay (ELISA)
For the quantification of TNF-α in the conditioned medium at each time point, sandwich TNF-α ELISA (KHC3012, TermoFisher Scientific, Carlsbad, CA, USA) was performed according to the manufacturer. Absorbance was read at 450 nm on a microplate reader (Synergy HT, BioTek, Winooski, VT, USA). A standard curve was built with concentrations ranging from 0 to 1000 pg/ml. The final concentrations were calculated by subtracting the value of each sample (i.e., testing conditions 1–3) to the conditioned culture medium baseline (i.e., testing condition 4), and the average was plotted for each testing condition along time.
2.6. Biological Assays
2.6.1. Isolation and Cell Culture
Cartilage tissue consists of only one cell type, the chondrocyte, embedded in a dense extracellular matrix (ECM). To evaluate the cytotoxicity of the developed biofunctionalized nanofibrous substrates, human articular chondrocytes (hAC) isolated from knee cartilage samples, collected from arthroplasties surgeries biopsies, were used. These cells were chosen as they were isolated from diseased joints cartilage. Samples were collected under the cooperation agreement between Centro Hospitalar do Alto Ave, Guimarães, Portugal, and the 3B’s Research Group, after informed donor consent. Briefly, cells were isolated by enzymatic digestion, according to the protocol described previously [46
Dulbecco’s modified Eagle’s medium (DMEM, Sigma D5671), containing 10 mM Hepes buffer (Life Technologies, Paisley, UK), l-alanyl-l-glutamine (Sigma), Non Essential Aminoacids (Sigma) 10,000 units/ml penicillin, 10,000 μg/mL streptomycin, and 10% foetal calf serum was the basis for the expansion and differentiation medium used herein (Basic medium). Basic medium was supplemented with 10 ng/mL of bFGF for hAC expansion. Basic medium was further supplemented with 1 mg/mL of insulin and 1 mg/mL of ascorbic acid, when hAC were seeded onto the PCL NFM (differentiation medium). These supplements enhanced extracellular matrix (ECM) deposition by the cultured hACs.
Human articular chondrocytes where used at passage 4. Expansion medium was changed every 2 days until the cells reached a confluence of 90%. The cells were harvested and seeded onto both the activated NFM, as well the NFM with the TNF-α antibody immobilized using the differentiation medium.
2.6.2. Seeding onto NFMs
Electrospun PCL NFMs were sterilized by UV-O treatment. All the surface functionalization and TNF-α antibody immobilization steps were performed in sterile conditions. For the biofunctionalized NFMs, antibody immobilization was performed overnight, and after the BSA blocking step, cell seeding was performed. Activated and biofunctionalized electrospun NFMs were used as controls.
Confluent hACs were detached from the culture flask using trypsin, counted on a hemocytometer and seeded at a density of 200.000 cells/NFM. Seeding was performed using the droplet method, using a 50 µL drop of cell suspension on all sample groups in 24-well plates. The plates were placed at 37 °C and 5% CO2 over 4 hours. After cell attachment to the NFMs, 1 mL of expansion medium was added to each well. Afterwards, the medium was changed into differentiation medium. The seeding was performed in three independent experiments. For each independent experiment, constructs were cultured for 1, 14, 21 and 28 days under static conditions and collected at each time point for quantification of cell viability, DNA, total protein analysis, as well as GAGs deposition. Cell morphology was evaluated by SEM.
2.6.3. DNA Quantification
DNA quantification was assessed using Quant-iT™ PicoGreen® dsDNA Reagent and Kit (Life Technologies, Eugene, OR, USA), according to the manufacturer´s instructions. Triplicates of each condition collected at 1, 14, 21 and 28 days were evaluated. The specimens were collected, 1 mL of sterile distilled water was added, and then samples were stored at −80 °C until further analysis. Prior quantification, the samples were defrosted and sonicated for 15 min. To extrapolate the DNA values for each sample, a set of standards were prepared with concentrations ranging from 0 to 1.5 μg/mL. The fluorescence of each sample was measured on an opaque 96-well plate using an excitation of 485/20 nm and an emission of 528/20 nm, using a microplate reader (Synergy HT, BioTek, Winooski, VT, USA).
2.6.4. Total Protein Synthesis Quantification
Total protein was performed using Micro BCA™ Protein Assay Kit (ThermoFisher Scientific, Rockford, IL, USA) according to the manufacturer´s instructions. Triplicates of each condition collected at 1, 14, 21, and 28 days were evaluated. The samples were collected as described in Section 2.6.3
. A standard curve was prepared ranging from 0 to 200 μg/mL. The absorbance of each sample was measured at 562 nm using a microplate reader (Synergy HT, BioTek, Winooski, VT, USA).
2.6.5. Glycosaminoglycan (GAG) Quantification
GAG quantification was performed using papain digestion. Triplicates of each condition collected at each previously established time point were tested. The samples were collected and stored in eppendorf tubes at −80 °C until further analysis. Digestion solution was prepared by adding papain (Sigma Aldrich, Saint Louis, MO, USA) and N-acetyl cysteine (Sigma Aldrich) at concentrations of 0.05% and 0.096%, respectively, to 50 mL of digestion buffer (200 mM of phosphate buffer containing 1 mM EDTA (Sigma Aldrich), pH 6.8). Each specimen was incubated with 600 μL of digestion buffer, overnight at 60 °C. After a 10-min centrifugation at 1300 rpm, the supernatant was collected. Dimethymethylene Blue (DMB) stock solution was prepared dissolving 16 mg of DMB powder in 900 mL of distilled water containing 3.04 g of glycine and 2.73 g of NaCl. pH was adjusted to 3.0 with HCl and the volume adjusted to 1 L. The solution was stored at RT covered by aluminum foil. Chondroitin sulphate (Sigma, C8529) solution was prepared in water in a 5 mg/mL stock solution and kept refrigerated. Standards were prepared from serial dilutions of this solution. Three samples per condition were considered per time point, and the absorbance of each sample was measured at 530 nm using a microplate reader.
2.6.6. Histological Analysis
Samples were collected at the end of the experiment and processed for histology. Samples were then fixed in 10% neutral-buffered formalin and then kept at 4 °C until the staining procedures. Alcian Blue staining was performed by rinsing the samples in 3% acetic acid, keeping them in 1% alcian blue solution (Sigma, A-3157) for 1 h. After that, the stain was poured off and sections were washed with water, let to dry, and then rinsed in absolute alcohol, cleared in xylene, and mounted in Entellan rapid (Merck Millipore, Darmstadt, Germany, 1.07960.0500).
2.7. Statistical Analysis
Statistical analysis of values related with antibody immobilization, TNF-α capturing profiles and biological studies was performed using IBM SPSS software (version 21; SPSS Inc., Armonk, NY, USA). Firstly, a Shapiro-Wilk test was used to ascertain the assumption of data normality. For antibody immobilization, protein capture and biological assays, the results showed that the data was not following a normal distribution. P values lower than 0.01 were considered statistically significant in the analysis of the results. A Kruskal-Wallis test was also performed for the comparison of more than two independent groups of samples for one variable.