Intervertebral disc degeneration is a common disease that can lead to axial skeletal pain, radiculopathy, and myelopathy. Combined physical and medical therapies are successful in relieving pain in approximately 90% of the cases. However, the remaining 10% become chronic and generate a serious public health problem, as chronic low-back pain (CLBP) ruins both the life quality and the labor capacity of the patient and increases the use of health services (1, 2).
Physical therapy and exercise are generally the first choice for treatment of CLBP. When these fail, several types of surgery are performed to relieve pain and decrease disability. The most common interventions are spinal fusion (arthrodesis) with damaged disc removal (discectomy) or substitution by artificial materials (arthroplasty; total disc replacement). The indication of surgery in CLBP is controversial because of its side effects, disturbance of motion and other biomechanical consequences that can accelerate the degenerative cascade at the operative level and at adjacent segments (3, 4). Despite of these risks, spinal fusion is the gold standard for CLBP (1, 5) and its analgesic value is beyond question. Figure 1 presents an extensive meta-analysis of seven high-quality recent clinical trials. Data on evolution of pain and discapacity were recalculated and expressed on a 0% to 100% scale. Quantification and comparison of several CLBP treatments was performed using the initial pain score versus pain relief plot (6) (for more details, see Table, SDC 1,http://links.lww.com/TP/A475).
The slope of the line defines the efficacy of any particular treatment, with complete pain relief attaining a slope of 1 (Fig. 1). The changes in pain scores (open symbols) and Oswestry Disability Indexes (ODI, closed symbols) can be plotted together, as the same relationship between the variables exists. Exercise, the conventional “nonsurgical” treatment (7), was the least potent treatment, with an efficacy of 0.07. The efficacy of treatments involving cognitive intervention plus exercise (8–10) ranged between 0.20 and 0.29, and spine fusion protocols were the most effective, with an efficacy ranging between 0.30 and 0.71 in different trials (7, 9–12). Disc replacement protocols with artificial discs had the same efficacy as fusion alone (11, 13). Finally, one trial (12) compared the effects of discectomy with discectomy plus treatment with cells expanded from the excised disc material (autologous disc cell transplantation in Fig. 1). A small but significant improvement was found in the group supplemented with cells after 2 years (see Table, SDC 1,http://links.lww.com/TP/A475) (12). However, a group with only cells was not included and thus, the effectiveness of these expanded cells alone is not yet known.
Progress in the understanding of degenerative disc disease pathophysiology has promoted study of new biologic therapies, including cell-based strategies. Potential advantages of these treatments are preservation of normal surrounding anatomy, biomechanics, and motion. Cell therapy has produced exciting results in both in vitro and in vivo (14), and studies with mesenchymal stem cells (MSC) have been particularly promising. Co-culture of MSC with nucleus pulposus cells stimulates both nucleus pulposus cells proliferation and MSC differentiation toward the chondrogenic lineage (15–18). Increased production of cytokines, particularly transforming growth factor-beta favors these transformations (18–20). The nucleus pulposus contains MSC that are similar to the MSC recovered from bone marrow (21), and studies in animal models of disc degeneration have shown that MSC injected in the nucleus pulposus area not only survive for months but also proliferate in canine (12, 22), porcine (23), and rabbit models (24). In addition, transplanted MSC induced production of extracellular matrix proteins, including aggrecan and other proteoglycans, and types I and II collagens (12, 23, 24). Finally, these studies also reported that injection of MSC resulted in better preservation of height and water content of the disc (12, 22, 25). Although animal data are very promising, MSC-based therapies have not yet been tested in humans. The only reported study involving treatment of CLBP with stem cells was carried out with unexpanded bone marrow hematopoietic precursors and no improvement of the pain was reported (26).
Based on the results of previous in vitro and animal experiments, we conducted a pilot study to test feasibility and safety and to obtain an early indication of the therapeutic value of MSC in human patients with chronic degenerative disc disease. Using autologous bone marrow Good Manufacturing Practice (GMP)-compliant MSC maximizes biosecurity of the protocol because of the previous experience with bone marrow transplantations. Furthermore, the intervention proposed here does not require surgery, does not produce anatomical modifications and does not hinder further interventions should they be required.
This pilot study included 10 patients (4 male and 6 female; average age 35±7 years) diagnosed of degenerative disc disease with preserved external annulus fibrous and persistent low-back pain. Additionally, all 10 patients did not respond to conservative treatment (physical and medical) lasting at least 6 months. Details on inclusion and exclusion criteria are presented in Supplemental Table 2 (see SDC 2,http://links.lww.com/TP/A476). The lesion was located at L4-L5 (2), L5-S1 (6), or both discs (2). Patients were recruited between June and December 2008 and treated from December 2008 to June of 2009. No major adverse events occurred.
The cell parameters were as follows (mean±standard deviation [SD]; n=10): bone marrow volume, 89±5 mL; total number of mononuclear cells obtained, 794±345×106; expansion time, 24±4 days; number of passages, 3; number of MSC released, 23±5×106; viability at the time of release, 87%±6%; viability at the time of application, 83%±5%. Expansion was performed under GMP conditions, with progression being monitored every other day. After 7 to 10 days in culture, cells became relatively homogeneous and had a fibroblastic appearance when approaching confluence. This morphology remained unchanged until release of cells for treatment (see Figure, SDC 3,http://links.lww.com/TP/A477). The antigenic profile conformed to the International Society for Cellular Therapy criteria for MSC (27) (see Figure, SDC 4,http://links.lww.com/TP/A478).
Evolution of Pain, Disability, and Life Quality
Table 1 summarizes the distribution of pain and disability indexes throughout the observation period. The starting point of pain and disability in the cohort was homogeneous. Patients on average felt intense lumbar pain (69±3 in the Visual Analog Scale [VAS]) and had moderate disability (ODI of 25±4) (mean±standard error [SE]; n=10). Six of the patients had sciatic pain at the beginning of the trial, and their evolution is also included in Table 1. On average, both lumbar pain and disability were strongly reduced at 3 months after MSC transplantation, followed by modest additional improvement at 6 and 12 months (Fig. 2A, B). Compared with the basal level of pain and disability, improvement was statistically significant at all time points (Fig. 2 legend). The sciatic pain followed the same trend, but the variation with respect to pain and disability among the patients was larger (Fig. 2C). The improvement in sciatic pain was significant at 6 and 12 months compared with the beginning of the trial. The pattern of improvement between VAS and ODI was parallel and resulted in global displacement of the whole distribution towards smaller values, with a strong decrease of the medians (P50% in Table 1). The improvements in pain and ODI at 12 months demonstrated a strong positive correlation with the values attained at 3 months (Fig. 2D; r=0.79; P<0.0001). The slope of the line was 1.17, indicating that the healing effect of MSC was rapid. In fact, 85% of the total improvement occurred during the first 3 months.
In Figure 3, we have plotted lumbar pain relief, assessed by VAS, at the end of the treatment as a function of the initial pain score (6) (Fig. 3; inverted triangles). The efficacy of the treatment is equal to the slope of the dotted line, which we compared to the “perfect treatment” with a slope of 1 (continuous line). The evolution of the disability index (circles) was plotted together with VAS-assessed values (inverted triangles) and exhibited the same relationship. Evolution of sciatic pain in the six affected patients was also followed (triangles). There was an excellent positive correlation between the initial score and the amount of improvement (r=0.78), indicating that the MSC treatment had a clear pain-relieving and disability-healing effect (P<0.0001). Regression analysis also resulted in a significant slope (P<0.0001). The slope of the line, which defines the efficacy of the treatment (6), was as high as 0.71, indicating that the effectiveness of MSC treatment is considerable. The results were consistently good for nine of the patients, but patient 3 (P3 in Fig. 3) did not demonstrate any apparent benefit from treatment.
The short form-36 (SF-36) life quality questionnaire revealed, by the end of treatment, a significant improvement of the physical component (summary value from 12.7±3.7 to 24.8±3.9; mean±SE; n=10; P<0.05) with no change of the mental component (from 54.1±10.6 to 49.7±10.5; P=0.77).
Magnetic resonance imaging (MRI) was used to assess disc height and water content of the discs. The heights of the affected disc segments were (in mm; mean±SE; n=10): 9.86±0.57, 9.81±0.55, and 9.84±0.63 at 0, 6, and 12 months of transplantation, respectively. These values were not significantly different. Water content of the discs, determined from T2-weighted sagittal images, was measured in the affected disc segment and in the contiguous 3 to 5 segments above the affected segment (see Figure, SDC 5,http://links.lww.com/TP/A479 and Methods, SDC 6, http://links.lww.com/TP/A480). A summary of the analysis of fluid content is shown in Table 2 and individual results are detailed in Supplemental Figure 3D (see SDC5,http://links.lww.com/TP/A479). The water content values for the affected disc are normalized by the values from the healthy discs in the same individual, which created a ratio. The ratio of fluid content of the affected segments to healthy segments was low at the beginning of treatment (mean±SE; n=11) at 0.62±0.03. This value did not change significantly at 6 months but increased to 0.72±0.03 at 12 months, a difference that was statistically significant (P<0.05, ANOVA; Bonferroni test; P=0.03, paired t test, two-tailed value).
Previous results from in vitro and animal studies indicated that using expanded bone marrow MSC to treat degenerative disc disease may be effective (14). This study, to the best of our knowledge, is the first to investigate this cell therapy in humans. Our results show that autologous MSC transplantation is both feasible and safe, with no major adverse effects recorded. The subjective evolution was favorable, and 9 of the 10 patients improved. The GMP-compliant cell preparation was satisfactory and production was reproducible with respect to number of cells (SD=22%) and expansion time (SD=17%). Immunophenotypic characteristics were also adequate and stable over time (see Figure, SDC 4,http://links.lww.com/TP/A478). Cell viability was good and not affected by transport to the site of administration or passage through spinal needles (data not shown). Quality control tests, including karyotype monitoring in three cell lots, were also satisfactory.
The analgesic effect of treatment with MSC was remarkable, approaching 71% efficacy. The improvement in pain was accompanied by a parallel improvement in disability (Fig. 3) and physical life quality (SF-36 results). In Figure 1, we have compared our results with the outcomes of previous clinical trials investigating other therapeutic interventions. Our results, labeled “This Study_MSC” in Figure 1, compare favorably with previous trials that explored physical treatments (7–10), and spinal fusion with or without disc replacement (7, 9–12) or complemented with expanded disc material (12). The analgesic effect of the intervention described here was rapid, as most of the improvement in pain (85%) was attained by 3 months (Fig. 2). Early action has also been reported for surgical interventions (12, 13).
In previous studies, no recovery of disc height has been reported 5 years after spinal fusion with or without total disc replacement (11) or 2 years after discectomy or discectomy plus cell treatment with cells expanded from the excised material (12). In the present study, we also found no improvement in disc height. However, the fluid content of the affected disc segments was significantly elevated at 1 year after the intervention (Table 2; see Figure, SDC 5,http://links.lww.com/TP/A479). This is consistent with the results obtained in animals, in which MSC were able to stop progression of disc dehydration and even induced water gain (22, 25). Injection of the excipient without cells had no effect (25). A slight increase of water content has also been reported in patients treated with surgery plus cells expanded from the excised disc, although this increase was seen only at 2 years after the intervention (12). We shall certainly follow the evolution of pain, disability, disc height, and disc water content in our patients in next year.
We can only speculate regarding the mechanism by which the beneficial effect of this treatment occurs. Animal studies have shown that MSC injected in the NP area are able to survive and proliferate (23, 24) and induce beneficial effects in degenerative disc disease (22, 25). Nucleus pulposus cells induce differentiation of co-cultured MSC into nucleus pulposus-like cells with a chondrocyte phenotype (15, 16, 19) and, even more importantly, MSC stimulated nucleus pulposus cells to proliferate and synthesize extracellular matrix (17, 18). This action may be important in vivo as few MSC are required to trigger this effect (18). In addition, MSC have a well-known immunomodulatory effect and express Fas-ligand when implanted in the spinal discs of dogs (22). These data indicate that MSC may help analgesia by reducing inflammation. Additionally, MSC can induce the production of anti-inflammatory cytokines (18). Because the analgesic effect is more evident than anatomical restoration, we may conclude that the trophic effects are faster than the regenerative ones, at least within 1 year after treatment. Note that, because of the potential systemic immune suppression and stromal support by MSC, concerns remain about possible facilitation of systemic infections and tumor growth. Long-term follow-up studies are required to address these issues (28).
Arthrodesis and arthroplasty restrict mobility and lead often to adjacent segment degeneration (1, 3). A continual decrease in disc height has been reported even after intervertebral disc transplantation (29). The alternative cell-based therapy proposed here avoids these side effects and is a simpler, less-invasive intervention. MSC treatment does not require surgery, and can be performed under an ambulatory regime. The cell treatment is expensive today, principally because cell production must be performed under astringent GMP conditions. However, the costs should decrease substantially in future as a result of larger scale production and more feasible regulation.
In summary, we propose that cell therapy with expanded bone marrow-derived mesenchymal cells should be considered as a putative treatment of chronic back pain caused by disc degeneration. Cell handling and expansion is reproducible and quality control tests were satisfactory. The clinical procedure is feasible and safe, and has several advantages over the current gold standard treatments: the intervention is simpler, more conservative, preserves normal biomechanics, does not require surgery or patient hospitalization, and results in the same or better pain relief. Future studies will involve larger trials centered in efficacy, with increased patient number and longer follow-up period. These studies will track the long-term evolution and investigate the anatomical and functional changes that occur in the intervertebral spaces. The possibility of cryopreserving a part of the harvested cells for subsequent multi-dose application would be extremely interesting to explore.
MATERIALS AND METHODS
Patients and Procedures
This study was designed as a pilot phase I trial. The protocol was approved first by the Teknon Medical Centre Ethics Committee and then by the Spanish Drug and Medicines Agency (EudraCT 2008-001191-68). Ten patients with chronic lumbar pain and positively diagnosed with lumbar disc degeneration and intact annulus fibrosus demonstrated by discography (30, 31) were included. Details on the inclusion and exclusion criteria are reported in Supplemental Table 2 (see Table, SDC2,http://links.lww.com/TP/A476). After complementary clinical, analytical, and imaging explorations to secure compliance with these criteria, the patient was informed about the characteristics of the protocol and, if he agreed to participate, he was asked to sign the informed consent.
The protocol included seven visits (V0–V6) with the following contents: V0, final check of compliance with I/E criteria, performance of all the needed complementary explorations and tests, programming of dates for V1 and V2; V1, bone marrow harvesting from the iliac crest (80–90 mL) for preparation of MSC. The patient was discharged after a 2-hr observation period. This intervention was performed under local anesthesia and slight sedation; V2 (3–4 weeks after V1), injection of MSC (10±5×106 cells per disc from a suspension containing 107 cells/mL) in the nucleus pulpous area of the affected segment (for details, see Methods, SDC 6,http://links.lww.com/TP/A480). V3–V6, are visits at 8 days, and 3, 6, and 12 months after implantation, which included clinical exploration and routine analysis (V3–V6), VAS, ODI, and SF-36 questionnaire, and quantitative MRI exploration (V5 and V6).
Cell Isolation and Expansion
Cell isolation and expansion were performed in the IBGM Cell Production Unit under GMP conditions and with specific approval of the Spanish Drug and Medicines Agency. The bone marrow sample was transported to the Cell Production Unit at 4 to 12°C within 12 hr of harvesting. The mononuclear cell fraction was isolated by density-gradient centrifugation, resuspended and cultured in the MSC expansion culture medium (21) in 175 cm2 tissue culture flasks with periodical washing to remove nonadherent cells. When cells reached 80% confluence they were trypsinized and replated, and the process was repeated for two more passages. At the end of this period (21–28 days), cells were harvested, resuspended in Ringer-lactate solution containing 0.5% human albumin (clinical grade, CSL Bhering GmbH, Marburg, Germany) and 5 mM glucose, and transported at 4 to 12°C by air courier (6 hr) to Teknon Medical Centre for application. In addition to quality control tests, viability and flow cytometric immunophenotypic profile (21, 27) were also determined at this stage.
In addition to the standard clinical and analytical procedures, the follow-up visits included measuring lumbar pain with VAS (6), disability with ODI, and life quality with SF-36 questionnaire (32). Results are expressed on a 0% to 100% scale in all the cases. MRI was used to measure disc height (22, 24) and to assess disc fluid content in T2-weighted sagittal images(see Figure, SDC 5,http://links.lww.com/TP/A479) (22, 25, 33).
Data are reported as mean±SD or SE, as indicated in each case. Significant differences were assessed using GraphPad Instat3 package software, GraphPad Software, La Jolla, CA.
The authors thank Mr. Jesús Fernández and Ms. Carmen Barbero for technical support. They also thank Dr. J.J. Velazquez, Traumatology Service, EGARSAT (Traumatology consultant), Dr. Andy Leist, Centro Médico Teknon (MRI), and Prof. J. Sentís, Department of Public Health, Medical School, University of Barcelona (advise with statistics).
1. Errico TJ, Gatchel RJ, Schofferman J, et al. A fair and balanced view of spine fusion surgery. Spine J
2004; 4: S129.
2. Balague F, Mannion AF, Pellise F, et al. Clinical update: Low back pain. Lancet
2007; 369: 726.
3. Harrop JS, Youssef JA, Maltenfort M, et al. Lumbar adjacent segment degeneration and disease after arthrodesis and total disc arthroplasty. Spine
2008; 33: 1701.
4. Rihn JA, Lawrence J, Gates C, et al. Adjacent segment disease after cervical spine fusion. Instr Course Lect
2009; 58: 747.
5. Kwon B, Katz JN, Kim DH, et al. A review of the 2001 Volvo Award winner in clinical studies: Lumbar fusion versus nonsurgical treatment for chronic low back pain: A multicenter randomized controlled trial from the Swedish lumbar spine study group. Spine
2006; 31: 245.
6. Huskisson EC. Measurement of pain. Lancet
1974; 2: 1127.
7. Fritzell P, Hagg O, Wessberg P, et al. 2001 Volvo Award Winner in Clinical Studies: Lumbar fusion versus nonsurgical treatment for chronic low back pain: A multicenter randomized controlled trial from the Swedish Lumbar Spine Study Group. Spine
2001; 26: 2521.
8. Brox JI, Reikeras O, Nygaard O, et al. Lumbar instrumented fusion compared with cognitive intervention and exercises in patients with chronic back pain after previous surgery for disc herniation: A prospective randomized controlled study. Pain
2006; 122: 145.
9. Brox JI, Sorensen R, Friis A, et al. Randomized clinical trial of lumbar instrumented fusion and cognitive intervention and exercises in patients with chronic low back pain and disc degeneration. Spine
2003; 28: 1913.
10. Fairbank J, Frost H, Wilson-MacDonald J, et al. Randomised controlled trial to compare surgical stabilisation of the lumbar spine with an intensive rehabilitation programme for patients with chronic low back pain: The MRC spine stabilisation trial. BMJ
2005; 330: 1233.
11. Guyer RD, McAfee PC, Banco RJ, et al. Prospective, randomized, multicenter Food and Drug Administration investigational device exemption study of lumbar total disc replacement with the CHARITE artificial disc versus lumbar fusion: Five-year follow-up. Spine J
2009; 9: 374.
12. Hohaus C, Ganey TM, Minkus Y, et al. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J
2008; 17(suppl 4): 492.
13. Siepe CJ, Tepass A, Hitzl W, et al. Dynamics of improvement following total lumbar disc replacement: Is the outcome predictable? Spine
2009; 34: 2579.
14. Fassett DR, Kurd MF, Vaccaro AR. Biologic solutions for degenerative disk disease. J Spinal Disord Tech
2009; 22: 297.
15. Le Maitre CL, Baird P, Freemont AJ, et al. An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue. Arthritis Res Ther
2009; 11: R20.
16. Vadala G, Studer RK, Sowa G, et al. Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion. Spine
2008; 33: 870.
17. Watanabe T, Sakai D, Yamamoto Y, et al. Human nucleus pulposus cells significantly enhanced biological properties in a coculture system with direct cell-to-cell contact with autologous mesenchymal stem cells. J Orthop Res
2009; 28: 623.
18. Yang SH, Wu CC, Shih TT, et al. In vitro study on interaction between human nucleus pulposus cells and mesenchymal stem cells through paracrine stimulation. Spine
2008; 33: 1951.
19. Risbud MV, Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: Implications for cell-based transplantation therapy. Spine
2004; 29: 2627.
20. Yang H, Wu J, Liu J, et al. Transplanted mesenchymal stem cells with pure fibrinous gelatin-transforming growth factor-beta1 decrease rabbit intervertebral disc degeneration. Spine J
2010; 10: 802.
21. Blanco JF, Graciani IF, Sanchez-Guijo FM, et al. Isolation and characterization of mesenchymal stromal cells from human degenerated nucleus pulposus: Comparison with bone marrow mesenchymal stromal cells from the same subjects. Spine
2010; 35: 2259.
22. Hiyama A, Mochida J, Iwashina T, et al. Transplantation of mesenchymal stem cells in a canine disc degeneration model. J Orthop Res
2008; 26: 589.
23. Henriksson HB, Svanvik T, Jonsson M, et al. Transplantation of human mesenchymal stems cells into intervertebral discs in a xenogeneic porcine model. Spine
2009; 34: 141.
24. Sakai D, Mochida J, Iwashina T, et al. Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model: Potential and limitations for stem cell therapy
in disc regeneration. Spine
2005; 30: 2379.
25. Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials
2006; 27: 335.
26. Haufe SM, Mork AR. Intradiscal injection of hematopoietic stem cells in an attempt to rejuvenate the intervertebral discs. Stem Cells Dev
2006; 15: 136.
27. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy
2006; 8: 315.
28. Bernardo ME, Locatelli F, Fibbe WE. Mesenchymal stromal cells. Ann N Y Acad Sci
2009; 1176: 101.
29. Ruan D, He Q, Ding Y, et al. Intervertebral disc transplantation in the treatment of degenerative spine disease: A preliminary study. Lancet
2007; 369: 993.
30. Adams MA, Dolan P, Hutton WC. The stages of disc degeneration as revealed by discograms. J Bone Joint Surg Br
1986; 68: 36.
31. Modic MT, Ross JS. Lumbar degenerative disk disease. Radiology
2007; 245: 43.
32. Davidson M, Keating JL. A comparison of five low back disability questionnaires: Reliability and responsiveness. Phys Ther
2002; 82: 8.
33. Watanabe A, Benneker LM, Boesch C, et al. Classification of intervertebral disk degeneration with axial T2 mapping. AJR Am J Roentgenol
2007; 189: 936.