Cell Isolation and Expansion
Bone marrow mesenchymal stromal cells, were obtained from 3 healthy donors that were subjected to autologous MSC transplantation and produced more cells than needed for their own treatment. Cells were processed using good manufacturing practice conditions in the IBGM Cell Production Unit as described previously.4,21 Isolations were carried out with the following parameters (mean ± SD; n = 3): BM volume = 103 ± 8 mL, number of mononuclear cells obtained = 1.1 ± 0.5 × 109, expansion time = 22 ± 2 days, number of MSC injected into the knee = 40 × 106 while suspended in Ringer-lactate at 5 × 106 cells/mL, and viability greater than 98%. A serum sample from each donor was obtained to screen for human immunodeficiency, hepatitis B, and hepatitis C virus by Nucleic Acid Amplification Technology.28 The cells obtained from each donor were used for 4 to 6 recipients (see Table 2 legend for details). Immune matching was not attempted.
Bone Marrow Harvesting, Purification, and Culture
Puncture and BM aspiration were performed in an ambulatory surgery session. The patient, in prone position, underwent light sedation. The surgical field was brushed with alcoholic povidone-iodine solution (chlorhexidine if a history of allergy to iodine exists) and delimited with sterile fields, leaving free both posterior iliac crests. After local anesthesia (20 mL of 1% lidocaine without epinephrine diluted v/v with saline), 2 members of the extractor team, placed on both sides of the operating table, performed several punctures with an 11-G trocar under the iliac spine, aiming toward the posterior sacroiliac joint (this is the iliac area with higher trabecular density). The technique involves sudden cortical perforation and repeated aspiration of small BM volumes (2-4 mL) to minimize contamination with the peripheral blood. The aspirate was injected into a heparinized bag for transport. Two successive aspirations were performed by rotating 90 degrees clockwise the beveled trocar. The same puncture hole allows a further 1 to 2 mm deepening twice, repeating the same methodology with 2 to 4 mL suction, syringe change, 90 degrees bezel rotation, and new aspiration. Then the trocar can be removed by sliding it slightly, and a few millimeters above the cortical pelvic, puncture can be repeated, continuing on both sides of the pelvis until about 80 mL are collected. Bone marrow (sterile bag heparinized with a volume of about 80 mL of aspirate) was refrigerated to 4°C, conditioned, and shipped to the Cell Therapy Unit. Further processing was done within 24 hours.
Donor serum samples were also obtained at this time to carry out the required screenings for excluding human immunodeficiency virus and hepatitis A and B contamination (Annex VIII and Directive 2004/23/EC of the European Parliament and of the Council). Tests must be performed by Nucleic Acid Amplification Testing to circumvent quarantine, which would otherwise be necessary to avoid the window effect.28 An aliquot of the serum sample must be stored to allow further analysis if required in future.
Bone marrow samples were transported to the cell production unit at 4 to 12°C within 24 hours of harvesting. The mononuclear cell fraction was isolated by density-gradient centrifugation, resuspended, and cultured in MSC expansion culture medium29 in 175-cm2 tissue culture flasks, with periodic washing to remove nonadherent cells. When cells reached 80% confluence, they were trypsinized and replated, and the process was repeated for 2 more passages. At the end of this period (21-24 days), cells were harvested, resuspended in Ringer lactate solution containing 0.5% human albumin (CSL Behring GmbH, Marburg, Germany) and 5 mM glucose, and transported at 4 to 20°C to the hospital application. In addition to quality-control tests, viability and flow cytometric immunophenotypic profiles29,30 were determined at this stage.
Magnetic resonance imaging was used to assess the cartilage state by T2 mapping using the GE CartiGram as described previously4 at baseline and 6 and 12 months after treatment. Briefly, mean T2 relaxation values (ms) were sampled in 88 well-defined regions of interest (ROIs) including at the patellar cartilage (24 ROIs), femoral condyles (32 ROIs), and tibial condyles (32 ROIs). We have shown previously that values in healthy individuals distribute normally, with a mean (and median) of 37 ms and a SD of 7 ms. The 95% percentile value was 50 ms.4 To analyze the assay results, we averaged the values in each area (88 ROIs). Those above 50 ms, which reveal poor-quality remodeling inflammatory tissue,31-33 were counted to compute the poor cartilage index (PCI, expressed as a % of all values obtained in the 88 ROIs), as described in the Results. Values above 90 ms were not used for computations. Values at 100% represent the worst possible PCI, and those at 5% or below are considered healthy. The MRI tests were completed in all the 15 control patients and 12 of the 15 test patients.
Data are reported as means ± SD or SE, as indicated. Significant differences were assessed by either Student t tests or by 1-way repeated-measures analyses of variance with appropriate corresponding nonparametric tests. We used GraphPad Instat3 package software version 3.06 (GraphPad Software, La Jolla, CA) for all calculations.
The 30 patients recruited for this study were given randomized treatments of either MSCs or hyaluronic acid. No serious adverse events occurred during treatment. Minor adverse events are summarized in Table 3. Transient mild local pain and discomfort in the injected knee with inflammation and swelling during the first 1 to 7 days occurred frequently in both the control and cell-treated patients (50-60% of patients). This pain was controlled with ibuprofen.
Cells obtained from the 3 healthy donors were relatively homogeneous and demonstrated a fibroblastic appearance when approaching confluence after 7 to 10 days in culture. This morphology remained unchanged until use. The antigenic profile of the cultures conformed to the International Society for Cellular Therapy criteria for MSCs.4,21,29,30
Evolution of Pain, Disability, and Quality of Life
Table 4 summarizes the distribution of knee pain and disability indices throughout the observation period. The starting point within the cohort ranged from mean values of 39 and 64 on a scale of 0 to 100 for the VAS, Lequesne index, and WOMAC index. Values of all evaluation scales improved more with the cell treatment than with the active control (hyaluronic acid). Improvement of patients following cell treatment was medium to large (effect size, 0.58 to 1.12 for the different algofunctional indices, Table 4), whereas improvement was small (effect size, 0.19 to 0.48) after hyaluronic acid treatment.
Pain was significantly reduced by 6 and 12 months after MSC transplantation (VAS and pain subscale of WOMAC, Figure 1A and B). The active controls treated with hyaluronic acid showed smaller improvements, not statistically significant except for the VAS value12 months after intervention (Figure 1A and B). The other algofunctional indices (general scale of WOMAC and Lequesne) changed consistently, with significant decreases at 6 and 12 months in the MSC-treated group and no significant change in the active controls (Figure 1C and D). The observed changes were parallel for the VAS, WOMAC, and Lequesne indices and resulted in displacement of the whole distribution toward smaller values, with decreased median values (P50%, Table 4). In the cell-treated group, 77% of the patients were satisfied or very satisfied with the treatment, whereas in the control group, this percentage fell to 38%.
Figure S2A (SDC, http://links.lww.com/TP/B133) shows knee pain relief at the 1-year follow-up, assessed by WOMAC (pain subscale), as a function of the initial pain score.24 Treatment efficacy is equal to the slope of the line, with a slope of 1 indicating the “perfect treatment.” Even though some scatter existed, a positive correlation was observed among values obtained in the MSC-treated group (r = 0.74; P < 0.02) around a line with a slope of 0.42 ± 0.10 (mean ± SE). In the controls treated with hyaluronic acid, the slope of the best-fit line was smaller, 0.10 ± 0.08. The difference between both slopes was statistically significant (P < 0.02), indicating that the treatment with cells was successful. The other indices reflected similar results, although the differences between the control and experimental groups were not always statistically significant, likely because of the dispersion of individual data. In Figure S2B (SDC, http://links.lww.com/TP/B133), we plotted the 3 main algofunctional indices: VAS, WOMAC (general), and Lequesne. We fitted a straight line to all control values on 1 side and to all experimental values on the other. This provided 2 lines with slopes of (mean ± SE) 0.14 ± 0.05 and 0.38 ± 0.06, respectively. The difference was highly significant (P < 0.005), confirming that the cell treatment relieved pain and disability.
In our previous study with autologous cells, we did not find an effect on the life quality, as measured by the SF-36 questionnaire.4 Consistent with these results, in the present study, we did not find significant differences between the control and the cell-treated patients in either the physical or mental component scores of the SF-12 questionnaire (Table S1, SDC, http://links.lww.com/TP/B133). However, the SF-36 questionnaire has been reported to be less sensitive for assessing knee arthritis than WOMAC, which was developed specifically for patients with lower extremity arthritis.35 In fact, in several previous studies, the SF-36 scores were barely modified in either control or treated osteoarthritis patients.36,37 Thus, we place more value on the WOMAC scoring system.
The MRI quantitative T2 mapping has been used previously to evaluate articular cartilage quality. T2 relaxation time is sensitive to both changes in cartilage hydration and collagen fibril orientation.32,38 T2 relaxation time is longer in remodeling inflammatory tissue versus hyaline cartilage32,33 and increases in osteoarthritis.38-40 Healing could then be followed by the decrease of the T2 relaxation time in affected areas. Figure 2A illustrates the decrease of T2 values in several areas of the articular cartilage after treatment with MSC in one of the patients. Note improvement after treatment in the areas indicated by white arrows. In order to perform a quantitative analysis, the articular cartilages were segmented in 88 ROIs (see details in Methods) and the mean T2 relaxations were computed. Figure 2B shows the transversal profile of an anteroposterior section and illustrates partial healing by treatment by MSC, with decrease of the mean T2 relaxation times in several ROIs.
In healthy knees, the mean T2 relaxation value of the 88 ROIs was (±SD) 37.0 ± 6.8 ms. Because 95% of values should be smaller than mean + 2 × SD, 50 (=37 + 2 × 6.8) was chosen as the threshold above which T2 values were considered inordinately high.4 For this reason, the PCI was computed as the percentage of T2 values greater than 50 ms. A PCI of 100 is the worst possible value and a value near 5 is considered healthy.4 Figure 3A compares the evolution of the PCI in cell-treated patients versus the active controls treated with hyaluronic acid. The PCI decreased in both groups, but the decrease was not statistically significant in the control, whereas it reached significance (P < 0.05) in the experimental group at the 12-month follow up. In Figure 3B, PCI improvement was plotted against the baseline PCI score. In the treated patients, the slope of the line (efficiency of treatment) was 0.69, whereas in the control series, the slope was only 0.28. However, the differences were not significant as dispersion of the data was rather high (DS = 0.49 and 0.38, respectively). Both lines intercepted the abscissa axis near the 5% value, which is the randomly expected value for nonaffected individuals (Figure 3B).
Previous studies have demonstrated that autologous MSCs provide excellent therapeutic alternative for treating osteoarthritis4-6; however, allogeneic cells, which have been extensively tested for safety (see Introduction) would be logistically more convenient. This trial was, to the best of our knowledge, the first to use allogeneic MSC to test therapeutic efficacy in knee osteoarthritis. Our results show that allogeneic MSC transplantation is safe and feasible and results in no major adverse outcomes. Postimplantation pain was observed in 53% to 60% of patients (Table 3) and showed similar frequencies in the experimental and control groups. This pain responded well to ibuprofen, vanishing within 1 to 6 days. This minor adverse outcome was also reported at a similar frequency (50%) in a trial that tested autologous MSCs.4
Improvement of cartilage quality can be followed by MRI through T2 mapping, which focuses on the evolution of poor cartilage areas and filters out most of the spurious variation, thus enhancing the sensitivity of the procedure.4 Cartilage healing can be conveniently followed by the decrease of the poor-quality cartilage areas, quantified by PCI. In our previous study, we showed that PCI improved significantly after treatment with autologous MSC and continued to improve through the second year follow-up.5 We find here that allogeneic MSC treatment also significantly improves PCI (Figure 3), suggesting that these MSC aid in cartilage repair or regeneration. Adipose tissue–derived MSCs have also been reported to aid in cartilage repair, as evidenced by MRI and histological indications of hyaline cartilage regeneration.6 Further investigation of the cartilage healing progression over a longer time period and investigation into the effect of repeated MSC application is warranted for future studies.
The analgesic effect of allogeneic MSC treatment is remarkable, resulting in 38% to 42% improvement in pain compared to 10% to 14% in active controls with hyaluronic acid (Figure 1A and B; Figure S2 A, SDC, http://links.lww.com/TP/B133). Improvements in the other algofunctional indices were similar (Figure 1C and D; Figure S2 B, SDC, http://links.lww.com/TP/B133). The effects were significant at 6 and 12 months for MSC-treated patients, whereas smaller improvements in the active control group were, in general, not statistically significant. The efficacy of the allogeneic MSC treatment for pain relief compares favorably to the reported for conventional treatments. For example, in a recent meta-analysis of 4 prestigious clinical trials, we find an efficacy range of 4% to 36 % for 8 different treatments (see Figure 3 in Orozco et al4). Table 5 compares the results obtained here with allogeneic cells to those of previous trials using autologous MSC. Efficacy was quantified for comparisons of different trials by dividing pain relief by the initial pain score.24 This gives values between 0 (no effect) and 1 (perfect treatment). Values of effect size, a primary measurement of significance (see Table 4 legend), are also shown. The efficacy of allogeneic treatment found in this trial was smaller than that reported for autologous BM -derived cells4 and similar to that reported with autologous adipose tissue-derived cells6 (Table 5); however, direct comparisons are difficult because the previous studies were uncontrolled. Apart from differences in the cell type, the fact that the present study was a multicenter endeavor that included two different surgical teams may have contributed to differences by increasing variability. In fact, it would be interesting to directly compare autologous with allogeneic cells in different arms of the same trial, and we are moving in this direction for future studies.
The results published here are the first to demonstrate feasibility and safety of allogeneic MSCs while providing strong indications for their efficacy in treating osteoarthritis. These results may contribute to more widespread use of MSC treatments because allogeneic cells are cheaper and more logistically convenient than autologous cells. Additionally, studies are increasingly demonstrating the biosafety of allogeneic MSCs (see Introduction), which also encourages clinical use. However, the transition from autologous to allogeneic MSCs should be made with extreme caution to ensure safety. Allogeneic MSC treatments will benefit from further research addressing how MSCs relieve pain, promote regeneration, and become immune evasive. In addition, protecting MSCs from immune detection and prolonging their persistence in vivo may improve clinical outcomes and prevent sensitization.8 Research that has been performed suggests that, due to the fast acting nature of MSC treatments, many of the early beneficial effects may be trophic by promoting immunomodulatory7,41 and/or anti-inflammatory effects.42 Importantly, MSCs have also been shown to stimulate cocultured cells to proliferate and synthesize extracellular matrix.43-45 In fact, transplanted MSCs engrafted into the joint are activated and express Indian hedgehog and other genes. These genes in turn promote expression of collagen II and other chondrogenic genes by host cells.46 Because of these hit and run effects, tracing MSC action may be elusive.
In summary, we propose that cell therapy with expanded allogeneic BM-MSC be considered as a treatment for chronic osteoarthritis. Cell handling and expansion of these cells is reproducible, and quality control tests are satisfactory. The clinical procedure is feasible and safe, and requires minimally invasive intervention without surgery or hospitalization. The procedure results in significant relief of pain and disability, and quantitative MRI evidence indicates partial articular cartilage healing. Advantages of allogeneic over autologous treatments include lower cost, higher homogeneity, and the possibility of using them in seropositive patients. The healing effects appear to be somewhat smaller than those reported for treatment with autologous MSC, but this should be confirmed in future studies designed to directly compare both cell types within the same trial.
The authors thank Mr. Jesús Fernández, Ms. Sandra Güemes, and Ms. Virginia Gordillo from IBGM, and Ms. Carmen Barbero from ITRT for technical support. Durolane (Bioventus; 60 mg of hyaluronic acid in a 3 mL) was generously provided by Dr. A. Esteras, Director of Medical Marketing, Zambon S.A.U., Spain.
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