Porcine articular chondrocytes were isolated previously from non-transgenic pigs according to previously published methods (7, 10), frozen at passages 0 and 1 in DMEM mixed with 10% DMSO and 50% FBS (both v/v) and stored in liquid nitrogen until use. PAC were thawed and expanded in DMEM with 10% FBS (v/v), 100 IU/ml penicillin–100 mg/ml streptomycin, and 25 µg/ml endothelial cell growth supplement (Millipore). All media and antibiotics were from Life Technologies and FBS from Biological Industries (Kibbutz Beit Haemek, Israel).

Mesenchymal stem cells were isolated from bone marrow of 14-day-old male Wistar rats (Animal Service, CHUAC) according to previously published procedures (23). The study protocols were approved by the Animal Ethical Committee of Galicia. Briefly, the ends of femurs were cut away, and bone marrow was extruded by flushing with 5 ml D-Hank’s solution supplemented with antibiotics. Marrow suspensions were dispersed by pipetting, filtered and centrifuged as previously described (23). Cell pellets from four rats were pooled in RPMI with 10% FBS and antibiotics, seeded and cultured in 100-cm² dishes (TM Nunclon). Adherent cells grown to 70% confluence were defined as passage-0 MSC and expanded for two passages for characterization before use (23).

Mesenchymal stem cells expressing firefly luciferase (LUC-MSC) were generated by lentiviral infection using Lenti-X™ Lentiviral expression System (Clontech). Viral supernatants were obtained from 293 T cells transfected with pGL 4.14-Luc2 (Promega) following standard procedures and stored at 4°C until transduction. MSC at 70% confluency were incubated sequentially with viral supernatants, washed and cultured for 2 days before selection with 1 µg/ml puromycin (Clontech) for 5 days. After selection, LUC-MSC were allowed to recover in complete media. Their function was tested in vitro by incubating the LUC-MSC and negative controls in phosphate-buffered saline (PBS) with 150 µg/ml of firefly D-luciferin (Biosynth AG, Staad, Switzerland) followed by bioluminescence imaging in an IVIS Lumina XR imaging system using Living Image 4.3.1 software (Caliper Life Sciences, Waltham, MA, USA).

Inducible nitric oxide synthase (iNOS) was detected by flow cytometry in MSC and murine fibroblasts (3T3L1) cultured normally or after exposure to 10 ng/ml lipopolysaccharides (Sigma-Aldrich) for 24 h. During harvest, cells were washed with PBS (MP Biomedicals), then fixed in 4% paraformaldehyde (w/v) (Sigma-Aldrich) for 10 min, washed twice, and pre-blocked with 2% (v/v) rat serum (Life Technologies). Cells were incubated with rabbit polyclonal antibody anti-iNOS (Abcam, Cambridge, UK) for 1 h, washed and incubated with goat anti-rabbit immunoglobulin-phycoerythrin (PE) (Dako, Glostrup, Denmark) for 1 h for measurements in a FACSAria using DIVA software (BD Biosciences).

Four different animal experiments were carried out that involved i.a. injection of PAC into the right femorotibial joint of Lewis rats (summarized in Figure S1 in Supplementary Material). The protocols were approved by the Ethics Committee of the University of Barcelona and the government of Catalonia. Experiments were performed following a group sequential design starting with a pilot with a low number of rats per group before proceeding with a larger study if considered appropriate (24). Male rats (weighing 200–250 g) were purchased from Harlan (Barcelona, Spain) and kept under controlled conditions of light and temperature with food and water ad libitum at the animal facilities of the University of Barcelona. Body weight and animal well-being were monitored throughout the study. In preparation for cell injection, PAC and MSC were washed thoroughly by centrifugation to remove the FBS, resuspended in DMEM with antibiotics (vehicle), and kept at room temperature. Cells were mixed with 15 µl of vehicle/joint for i.a. injections, 200 μl/rat for intraperitoneal (i.p.) injections, and 100 μl/rat for intravenous (i.v.) injections. All injections were performed in animals anesthetized with isofluorane.

For the MSC localization studies (Figure S1A in Supplementary Material), we used a total of 18 rats distributed in six experimental cohorts (n = 3/group). Control rats received a single injection with 2.5 × 10⁶ cells/kg body weight of LUC-MSC i.a., i.v., or i.p. The other rats were subjected to one of three different regimes involving pretreatment: (1) one cohort received DMEM i.a. followed by i.a. injection of LUC-MSC (2.5 × 10⁶ cells/kg body weight) 2.5-weeks later, (2) and (3) the others were injected i.a. with 3 × 10⁶ PAC and 2.5 weeks later with the described amount of LUC-MSC, either i.v. or i.p. The follow-up by bioluminescence imaging was established at 2 and 24 h, and at 4 and 5 days after MSC injection in an IVIS Lumina XR imaging system using Living Image 4.3.1 software. For each determination, the rat was injected i.p. with 200 mg/kg of firefly D-luciferin and 15 min later was analyzed in the IVIS chamber under anesthesia with isofluorane.

In a pilot experiment assessing the immune response (Figure S1B in Supplementary Material), six male rats were equally distributed between two experimental groups to receive either PAC i.a. (cohort PAC, n = 3) or MSC i.v. and PAC i.a. (cohort MSC + PAC, n = 3). Injection of 3 × 10⁶ PAC was considered time 0, whereas MSC (2.5 × 10⁶ cells/kg body weight) were injected i.v. 1 week before the PAC. The serum antibody titers were monitored at 2, 5, 10, and 15 weeks. Cellular responses of isolated peripheral blood lymphocytes (PBL) were determined by coculture assay at 5 weeks post-PAC injection. One rat of the MSC + PAC group died after week 11. The loss was preceded by an arrest in growth that was detected at week 10, whereas the rest of rats continued to grow throughout the study.

In another experiment (Figure S1C in Supplementary Material), 22 rats were distributed between four experimental groups to receive either DMEM i.a. (cohort DMEM, n = 5), PAC i.a. (cohort PAC, n = 6), MSC i.p. (cohort MSC, n = 5) or PAC i.a. and MSC i.p. (cohort PAC + MSC, n = 6). Injection of 3 × 10⁶ PAC was considered time 0, whereas MSC (2.5 × 10⁶ cells/kg body weight) were injected 3 weeks later. The immune response was monitored at 2, 5, 10, and 18 weeks after PAC injection by measurement of serum anti-PAC antibody titers and cytokine profile at 2 and 5 weeks. Body weight was measured at the various time points. When indicated, one additional milliliter of blood was obtained from each of the five rats/cohort used for isolation of PBL and conducting coculture assays. For follow-up of the anti-MSC antibody response, the time points were adapted to consider the time of MSC injection as time 0, and the 5, 10, and 18 weeks post-PAC injection became, respectively, 2, 7, and 15 weeks after MSC injection.

An additional separate study was performed with 18 rats to assess the immune response at the level of draining lymph nodes (Figure S1D in Supplementary Material), Rats were equally distributed between experimental groups to receive either DMEM i.a. (n = 6) and 3 × 10⁶ PAC i.a. (n = 6) or MSC i.v. (2.5 × 10⁶ cells/kg body weight) 1 week before 3 × 10⁶ PAC i.a. (n = 6). Two weeks after the injection, the popliteal lymph nodes were harvested for lymphocyte isolation.

The antibody response was assessed by flow cytometry using PAC or MSC as target cells and rat serum diluted as indicated for reactivity measurements. After incubation for 30 min at 4°C, anti-rat secondary antibodies [PE-conjugated goat F(ab′)2 anti-rat IgM and FITC-conjugated goat F(ab′)2 anti-rat IgG (Beckman Coulter Inc., Fullerton, CA, USA)] were added for further incubation at 4°C. In a separate set of experiments, PAC were incubated with the same sera (at 1%) up to week 10 to detect the different anti-PAC IgG subtypes using anti-rat secondary antibodies [mouse anti-rat IgG1, IgG2a, IgG2b, and IgG2c mAbs-FITC (Southern Biotech Inc., Birmingham, AL, USA)]. In both cases, cells were finally washed with PBS and analyzed by flow cytometry (Beckman Coulter Gallios™, Kaluza^® Analysis Software).

Peripheral blood lymphocytes were isolated from 1.5 ml of rat blood by Ficoll gradient followed by 2-h incubation at 37°C in RPMI/10% FBS to remove adherent cells. For isolation from lymph nodes, these were submitted to mechanical disruption into PBS/10% FBS and left to settle to remove large debris. Supernatants were transferred to clean tubes, washed by centrifugation and cell pellets resuspended in RPMI/10% FBS. In both cases, supernatants were recovered for cell counting in a hemocytometer in preparation for the assays.

T-cell activation was determined for individual rats in cocultures adapted from previously reported techniques (8, 25). Briefly, confluent PAC were incubated at 37°C in 96-well plates with PBL at 10:1 effector/target ratio. Controls of each cell type cultured alone were included. For additional activation, concanavalin A (ConA, Sigma) was added to selected wells at a final concentration of 10 µg/ml 30 min after initiating the coculture. Culture supernatants were harvested 24 h later, and IL-2 secretion was measured using Rat IL-2 ELISA kit (RayBiotech, Inc., Norcross, GA, USA).

For characterization, isolated lymphocytes were stained with anti-rat CD4-FITC and CD8-FITC antibodies alone or in combination with APC-conjugated anti-mouse Ki-67, PE-conjugated anti-Foxp3 or isotype control (all Miltenyi Biotec) for two-color analysis. The FoxP3 Staining Buffer Set (Miltenyi) was used for permeabilization and fixation. Cell surface staining of a gated population of lymphocytes was then measured in a Gallios flow cytometer.

Serum levels of 34 protein markers (Table 1) were assessed for most rats at baseline, week 2 and 5 postinjection by antibody array (Rat Cytokine Antibody Array G2, RayBiotech) with the technical support of tebu-bio (Le Perray-en-Yvelines, France). Final data corresponded to the average intensity of duplicate spots with the system’s background subtracted and normalized using internal controls.

Data are presented as mean and SEM. Non-parametric statistical tests were used due to non-normal distribution of data. Statistical analysis was performed with the Statistical Package for Social Sciences (version 12.0, SPSS Inc., IL, USA) using Wilcoxon rank-sum test or the Mann–Whitney U test (called Mann–Whitney U thereafter) when comparing two groups of independent samples and Wilcoxon signed-rank test (called Wilcoxon thereafter) for the comparison between follow-up and baseline values. Differences were considered statistically significant at p ≤ 0.05.

The distribution of allogeneic MSC in Lewis rats previously injected i.a. with PAC was firstly studied (Figure S1A in Supplementary Material). To this purpose, bone marrow-derived MSC isolated from Wistar rats were genetically modified to express luciferase. These LUC-MSC, but not unmodified MSC, produced good bioluminescence signals when assayed in vitro, which confirmed activity in the presence of luciferin (Figure 1A). In vivo, the biodistribution was highly dependent on the injection route (Figure 1B). Control rats (without PAC i.a.) injected with LUC-MSC i.a. showed photon signals localized at the injection site for up to 5 days, although with reduced signal over time. No diffusion of the signal was observed for any of the rats in this group that would indicate leakage from the injection site for the first 24 h. In the other cohorts shown in Figure 1B, injection of PAC i.a. before MSC did not lead to MSC recruitment into the joint. The i.v. route led to MSC entrapment in the lungs with predominance of one lung over the other. The bioluminescence intensity was reduced by 24 h in all cases in this cohort and disappeared completely by day 4. On the contrary, MSC administered i.p. remained alive longer and were detected in the abdomen during the 5-day study period. Notably, no photon signals were observed at the joint area at any time point or animal following the i.v. or i.p. routes. Rats receiving MSC via these routes without prior PAC injection displayed an identical pattern (data not shown).

As a major goal in our study, we sought to assess whether i.a. injection of PAC induced an immune response. As a second objective, additional experimental groups were included to study the effect of MSC systemic administration. The MSC utilized were confirmed to express iNOS at higher proportion than fibroblasts in stimulated conditions (Figure S2A in Supplementary Material). In an initial pilot experiment, we compared the immune response induced in a small number of rats either injected i.a. with PAC only or pretreated with MSC i.v. 1 week before the PAC injection (MSC + PAC cohort) (Figures S1B and 2B in Supplementary Material), An elicited anti-PAC IgM and IgG response was observed in all rats after i.a. injection of PAC (Figure 2). However, pretreatment with MSC i.v. did not reduce the antibody titers. In fact, the IgG antibody response may have been enhanced in MSC + PAC rats (Figure 2B). Likewise, MSC pretreatment seamed to increase overall T-cell reactivity in a coculture assay conducted to assess the cellular response using PAC and PBL isolated from each rat (Table 1). Although the low sample size did not allow statistical analyses for confirmation of these preliminary results, we chose to stop the experiment to avoid using more animals with these potential undesired effects.

The main experiment was designed to study the immune response to PAC-injected i.a. compared with a cohort receiving vehicle alone (DMEM), as well as the effect of MSC on this response once initiated (Figure S1C in Supplementary Material). To this end, MSC were administered i.p. 3 weeks after PAC injection in a PAC + MSC cohort. Notably, no deleterious effects were observed for any of the treatments (PAC or MSC) on the well-being and body weight of the animals throughout this experiment (Figure S2C in Supplementary Material). To monitor the humoral response, anti-PAC antibody titers were determined in all cohorts up to 18 weeks. Anti-MSC antibody reactivity was also measured for the DMEM and MSC cohorts. The cellular response was studied by assessing the serum cytokine/chemokine profile and setting up coculture assays with PBL isolated from each rat at selected time points.

Notably, no anti-MSC antibody (IgM or IgG) reactivity was detected in sera of rats injected with MSC over controls for the duration of the study (up to 15 weeks after MSC injection) (Figure 3).

Anti-PAC IgM and IgG responses were detected by flow cytometry in all rats injected with PAC but not in controls (DMEM and MSC cohorts) (Figure 4). Both anti-PAC IgM and IgG showed the highest reactivity at week 2 (first measurement after baseline) and diminished slowly afterward, although remained above baseline levels by week 18. No effect of the MSC (injected at week 3) was observed on the antibody response (Figures 4C,F). The elicited antibodies were further characterized in the cohorts injected with PAC i.a. by measuring the anti-PAC IgG subtypes (Figure 5). The profiles for rats injected with or without MSC were identical. The four IgG subtypes increased after PAC injection, although with different patterns. Changes in anti-PAC IgG1 and IgG2c followed the same timing of transient elevations at week 2 followed by dramatic decreases afterward. On the contrary, anti-PAC IgG2a peaked at 5 weeks and remained high at 10 weeks, whereas anti-PAC IgG2b remained high after the first increase at week 2.

Serum levels of 34 proteins were assessed by antibody array (Table 2) in 32 samples from individual rats of the DMEM, PAC, and PAC + MSC cohorts including at baseline and at 2 and 5 weeks. No differences were observed for most proteins tested, but results corresponding to 12 markers associated with inflammation and T-cell responses (activin A, fractalkine, GM-CSF, ICAM-1, IFN-γ, IL-1α, IL-2, IL-4, IL-10, L-selectin, MIP-3α, and TIMP-1) seemed potentially modified by the treatments (Figure 6) and were further evaluated. Thus, data from the PAC and PAC + MSC cohorts at baseline and week 2 were combined because an identical protocol was applied before week 3. Results and statistical analyses of the data pooled (n = 8) and shown for 10 of these proteins in Table 3 allowed to appreciate a distinct pattern between the DMEM group and the cohorts receiving PAC. Whereas most of these selected markers remained unchanged or even diminished in the vehicle-injected cohort, there was a profile associated with increases in serum marker levels after PAC injection at week 2 (Figure 6; Table 3). By week 5 postinjection, all these proteins had returned to baseline levels with exception of fractalkine (Figure 6). IL-2 and L-selectin were significantly elevated 2 weeks after PAC injection (p = 0.036, n = 8). Nonetheless, IL-1α was also significantly elevated, whereas GM-CSF and ICAM-1 showed similar trends toward significance. The effect of MSC on the markers was less clear, but it was still of interest to compare the profiles of the PAC and PAC + MSC cohorts at 2 and 5 weeks to assess the presence of some counteractive trends (Figure 6).

Coculture assays were set with PAC and PBL isolated from these rats at 2 and 10 weeks postinjection for IL-2 measurements (Table 4). This type of coculture provided information on the level of T-cell activation and co-stimulatory capacity (with ConA) in the presence and absence of PAC. The data showed for most rats and time points that there was some T-cell reactivity after coculture with PAC, which was further enhanced in the presence of ConA. Interestingly, the amount of IL-2 secreted in coculture was lower for PBL from PAC-injected rats than in those not exposed to PAC, although the co-stimulatory signals were preserved (elevations with ConA). Moreover, rats injected with MSC alone showed a profile very similar to the PAC cohort. Nevertheless, no major differences were observed between the PAC and PAC + MSC cohort at this level that could indicate an additional beneficial effect provided by MSC injected 3 weeks after PAC (Table 4, week 10). Very similar results were obtained when we analyzed PBL from three rats collected at week 5 of the experiment (data not shown).

In a final study, rats received either DMEM i.a., PAC i.a. or MSC i.v. 1 week before PAC i.a. to assess the cellular immune response at the level of draining lymph nodes 2 weeks after injection (Figure S1D in Supplementary Material). The mean (± SEM) number of total lymphocytes after isolation were, respectively, 2.7 ± 0.37 and 2.87 ± 0.35 million for the PAC and MSC + PAC cohorts versus 0.98 ± 0.07 million for the DMEM cohort (p = 0.004 for both). Flow cytometric analysis showed that the percentage of CD4⁺ and CD8⁺ lymphocytes remained within the same range for all cohorts (Figure 7A), although CD4⁺ cells showed a higher proliferation as detected by Ki-67 staining 2 weeks after PAC injection (Figures 7B,C). The percentage of Foxp3 cells was not clearly increased after PAC injection (Figure 7C), although most rats pretreated with MSC had higher numbers of these cells (Figure 7C).