A randomized, double blind, controlled study of adult human. mesenchymal stem cells delivered via intra-articular injection to the

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1 A randomized, double blind, controlled study of adult human mesenchymal stem cells delivered via intra-articular injection to the knee joint following meniscectomy Authors/Study Investigators C. Thomas Vangsness Jr., MD David Fox, MD David Dellaero, MD David Griffin, MD Jack Farr, MD Joel Boyd, MD John O Donnell, MD University of Southern California Orthopaedic Surgery Associates Unlimited Research Triangle Orthopaedic Associates, PA Orthopaedic Center of Vero Beach OrthoIndy TRIA Orthopaedic Center Greater Chesapeake Orthopaedic Associates, LLC 1

2 Abstract Introduction: In a double blind, controlled study, the safety and exploratory efficacy of an allogeneic mesenchymal stem cell (MSC) drug injection was investigated in patients after medial meniscectomy surgery. The purpose was to evaluate the effect of an MSC injection on meniscus regeneration and osteoarthritis (OA) progression. Methods: 55 patients at 7 institutions underwent a randomized, prospective, doubleblind trial. Under IRB approval, a single superior lateral knee injection was given 7-10 days after meniscectomy and aspiration of the knee. Patients were randomized to one of three single injection groups: low dose (50 million MSCs), high dose (150 million MSCs) and a hyaluronic acid (HA) vehicle control. Patients were followed for safety, pain, meniscus regeneration and overall knee joint condition. Timed intervals out to 2 years evaluated these parameters along with sequential MRI evaluations reviewed by a team of independent blinded musculoskeletal radiologists. Results: In the two MSC treatment groups, there were patients who exhibited increased meniscal volume (reaching the 15% predefined threshold based on MRI quantitative analysis, 23.5% in the 50 million cells group and 6% in the 150 million cells group) at the 12 month time point. There were no patients in the vehicle control group who exhibited the minimum 15% volume increase in the meniscus. Patients with osteoarthritic changes in their joint receiving MSCs experienced a statistically significant reduction in pain compared to those receiving the control, based in visual analog scale assessments. Recipients receiving the control were 3.5 times more likely to experience degenerative bone changes associated with OA as compared to those receiving MSCs 2

3 at 2 years. The OA effects were dose dependent and pain scores improved following MSC treatment. Discussion and conclusion: The single MSC injections were found to be well tolerated at 2 years following surgery with no ectopic tissue formation. Meniscus regeneration was statistically significant at the 12 month timepoint but not at 6 months and 2 years. An improvement in knee pain was noted in the MSC group suggesting a decrease in knee OA progression and better joint condition with MSC treatment. Key words: Articular Cartilage, Knee Arthroscopy, Meniscal repair, Meniscal Transplants, Meniscus, MRI, Mesenchymal Stem Cells 3

4 Background Arthroscopy of the knee is one of the most common surgical procedures today. These procedures are often to treat soft tissue injury of cartilage or the meniscus. It is estimated that 1.3 million meniscal pathology-associated procedures were performed in the United States in 2005 (Datamonitor 2000). Injuries to the meniscus or other soft tissue structures such as the ligaments of the knee often lead to degeneration of the joint and progression to osteoarthritis (OA) (Gillquist 1999). Isolated meniscus tears and subsequent repair increase the risk of OA 10-fold (15%-20% incidence) and meniscectomy in a joint with intact ligaments further doubles the risk (30%-40% incidence). OA is the most prevalent form of arthritis and is the leading cause of chronic disability, functional loss, and morbidity in the United States (Dunlop 2003). OA is a degenerative joint disease characterized by the progressive deterioration of cartilage with subsequent joint space narrowing, osteophyte formation, and subchondral sclerosis (Kijowski 2006). These structural changes can manifest clinically as joint pain, stiffness, swelling, and limited mobility. Symptomatic OA of the knee impairs an individual s ability to stand, walk, climb, and engage in other physical activities, thereby reducing overall quality of life. There are few options apart from surgical intervention for patients with knee disorders, and the available therapies for pain are not disease-modifying therapies. Mesenchymal stem cells are cells of mesodermal origin that have the capacity to differentiate into connective tissues such as bone, cartilage, tendon, ligament and fat (Pittenger 1999). They also have been observed to have potent anti-inflammatory and immune 4

5 modulatory effects (Aggarwal, Newman). In a series of preclinical meniscectomy animal studies MSCs were shown to have a potential for neomeniscal tissue formation and joint preservation (Murphy 2003, Murphy 2005). Mechanical instability was induced in the knee joint of goats by surgical resection of the anterior cruciate ligament and/or meniscus. Subsequently, MSCs were delivered to the joint by direct intra-articular injection. The injected cells became associated with the injured connective tissues. Safety and retention studies demonstrated that MSCs were concentrated on soft tissues within the joint and viable at 7 days post injection. Twelve-month safety studies in the normal knee indicated no significant negative findings on pathological or histological examination. Based upon preclinical studies in goats, it was believed that a single injection of hmscs delivered to the joint intra-articularly could initiate meniscal regeneration with the potential for long-term chondroprotection. Substantial evidence now exists that preparations of these cells can be delivered allogeneically without adverse immune effects (Kebriaei 2009, Hare 2009). Allogeneic therapy allows for immediate treatment in acute settings with a good source of cells that have been well qualified. Together, these findings led to a clinical investigation of MSCs as a potential treatment option following injury to the knee. The goal was to prevent the expected consequences of partial meniscectomy surgery, including increasing meniscal tissue formation and improving overall joint condition. The objectives were to evaluate the safety of intraarticular injection of MSCs to the knee joint, their ability to promote meniscus repair following surgery and limit changes characteristic of OA to the joint. 5

6 Methods Trial Design and Disposition This trial was a phase I/II, multicenter, randomized, controlled, double-blind, multipledose study of MSCs delivered intra-articularly after meniscectomy. Sixty male or female patients 18 to 60 years old, inclusive, meeting the main criteria for inclusion were randomly assigned to each of the 3 treatment groups (50 million hmscs-group 1, 150 million hmscs-group 2, or vehicle control-control). The trial was conducted in compliance with current Good Clinical Practice (cgcp) standards and in accordance with the principles set forth under the Declaration of Helsinki (1989). Institutional Review Board (IRB) approval of the treatment protocol was obtained at all centers prior to the initiation of patient enrollment. All patients entering the trial agreed to and signed an IRB approved statement of informed consent. Patients who required partial medial meniscectomy and were otherwise healthy were enrolled in the study. A total of 55 were treated: 18 patients were in Group 1(50 million hmscs), 18 patients were in Group 2 (150 million hmscs), and 19 patients received control. A total of 8 patients were discontinued from the study, of which 5 were discontinued before treatment with the investigational agent. Patients received the treatment injection 1 week after meniscectomy and were assessed for treatment safety and efficacy at different time points during the 2-year study duration. Fifty-four patients completed the study through 1 year and 52 patients completed the study visits through 2 years. 6

7 Composition of Active Investigational Agent and Control The active investigational agent was human mesenchymal stem cells, hmscs, isolated from bone marrow aspirates obtained from donors that are unrelated and not HLA matched to recipients. All donors are tested according to the FDA Donor Suitability Guidance prior to donation. The phenotype of the hmsc population was characterized by a cell surface profile of CD105+, CD166+, and CD45. The formulated treatment injection consisted of 1 injection of either 50 million or 150 million donor-derived hmscs suspended in 5 ml of diluted sodium hyaluronan or 1 injection of vehicle control delivered 7-10 days after meniscectomy. The active agent was composed of donor-derived hmscs suspended in 5 ml of sodium hyaluronan (20 mg)/plasmalyte/human serum albumin (1.2%). The vehicle control was composed of 5-mL sodium hyaluronan (20 mg)/plasmalyte/human serum albumin (1.2%). Release testing of the cells included measurement of cell-surface markers CD105 and CD166, absence of CD45, and karyotyping to exclude chromosomal abnormalities. (Osiris Therapeutics, Inc., Columbia, MD), a highly purified preparation of ex-vivo cultured adult hmscs. Patient Population Diagnosis for inclusion was made during the arthroscopic procedure (partial medial meniscectomy surgery). To be eligible, patients must have undergone partial medial meniscectomy requiring removal of at least 50% of the meniscus. The affected joint was required to have normal axial alignment. Any previous ligament construction must have been determined to the stable. Patients had to be willing to follow standard 7

8 postoperative rehabilitation and to participate in follow-up for 2 years from the time of meniscectomy surgery. Exclusion criteria of interest included confirmation at surgery of anterior cruciate ligament injury other support structure damage or Grade 3 or 4 cartilage damage. Also, if a patient had hyaluronic acid, steroid, or corticosteroid injections in the preceding 3 months, diffuse synovitis at the time of arthroscopy, or inflammatory arthritis, they were deemed ineligible. Patient enrollment and randomization Patients were enrolled by study investigators once they confirmed eligibility. When it was established that the patient met all inclusion criteria, randomization was performed. Randomization was conducted in a centralized manner. Manual randomization was performed using sealed envelopes at a 1:1:1 distribution across the three treatment groups. Prior and Concomitant Therapy Concomitant medications were recorded continuously during and after the investigational agent injection. All medications were coded according to preferred terms using the World Health Organization drug dictionary. Concomitant medications were summarized by each preferred term for the safety population (ie, all randomly assigned patients who received any study therapy). In the first 6 months of the study evaluation period, the following were not permitted by the study: corticosteroid injections, hyaluronic acid injections other than those prescribed by the protocol, oral glucosamine/chondroitin or oral corticosteroids. In addition, patients were required to 8

9 avoid running and/or repetitive impact activity during the first 6 weeks after surgery and strenuous activities or prolonged weight bearing for 48 hours after study injection. Safety - Clinical Assessments Physicians and other clinical personnel remained blinded to treatment assignment for all patients throughout the study period. Primary safety assessments included monitoring and recording all adverse events (AEs), and serious adverse events (SAEs). Safety laboratory and urine values, regular vital sign measurements and physical examination results were recorded as well. In addition to a standard physical examination covering major systems, an examination of the knee was performed to inspect for redness, swelling, deformity, abnormal tissue presentation and/or skin changes. The MRI images were reviewed for any signs of abnormal tissue formation (ectopic tissue) inside the joint capsule and in the surrounding tissues. The images were also reviewed for any abnormal bony changes. The expression of various immune cell markers for T cells, natural killer cells, and B cells in the peripheral blood was analyzed to detect general changes in the immune system relative to Baseline (preoperative screening). This provided an indication of any cell-mediated or humoral immune response to the investigational agent. Safety - Adverse Events Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events. The AEs were graded on a 5-point intensity scale ranging from 1 (mild) to 5 (death). The assessment of severity was made independent of the relationship to investigational agent administration or the 9

10 seriousness of the event. The relationship of an AE to investigational agent was assessed by the investigator. Magnetic Resonance Imaging Magnetic resonance imaging was performed on a clinical grade unit using a transmit/receive, phased array knee coil. A cartilage-sensitive pulse sequence using moderate cho time, fast spin-echo techniques was performed, which accentuated inherent magnetization transfer, yielding differential contrast between fluid, articular cartilage, and subchondral bone/calcified cartilage. In addition, the normal meniscus appeared hypointense on this pulse sequence because of its highly ordered structure that yielded relative restriction of water. This was effective in differentiating regenerated meniscal tissue from the native remnant. Preoperative MRIs were assessed for cartilage degeneration. The subchondral bone and plate were also assessed for areas of thickening and sclerosis. Meniscal changes were monitored with MRI throughout the study. Magnetic resonance imaging was performed as a preoperative screening measure (within 30 days before the surgery); after the meniscectomy at Visit 1 (baseline, before injection, Visit 2 (6 weeks), Visit 4 (6 months), Visit 5 (12 months), and Visit 7 (2 years). The meniscus was assessed for the size of the remnant (as judged from the zone of resection), the signal characteristics of the remnant (which reflected the degree of degeneration), and fraying of the inner edge. At the before injection and follow-up MRIs, all of the previously mentioned criteria for joint changes were assessed, and any additional alterations in signal intensity and size of the meniscal remnant were measured. 10

11 Field of view (FOV), matrix, and slice thickness constant throughout the examination and on subsequent visits. Scans were performed in the axial plane, sagittal plane, and coronal plane. The axial plane scans had contiguous coverage from above patella to below the tibial tubercle. The sagittal plane scans were aligned parallel to the lateral femoral condyle covering the entire knee. The coronal plane scans were perpendicular to the lateral femoral condyle and also covered the entire knee. Computational analysis of meniscal volume was performed by two independent university based musculosketal radiologists. To assure blinding in the early stages of the study, random knee images were instated in the sequence of the analyses. Slice-omatic v 4.3 software (TomoVision, 4559 Pontiac, Montreal, Quebec, CANADA, H2J 2T2) was used to digitize the DICOM images. Images of each 2 mm slice were digitized and the 3D volume reconstructed in each plane of MR image acquisition. Meniscal boundaries of each slice will be determined in two consecutive steps. First, the outline of the body of the meniscus was determined though automatic signal analysis using threshold segmentation. This was followed by visual inspection of signal intensity of border pixels in step two. Each border pixel was individually screened using the threshold segmentation mode and either included or excluded from the meniscal volume. Pixel size, determined by the FOV and resolution, was 0.42 x 0.42 mm. A qualitative analysis of the meniscal remnant and any new tissue that was present, as well as analysis of the cartilage and joint structures was performed. Changes in the extent of cartilage degeneration, osteophyte formation, and joint edema were tracked. The joint was examined for evidence of ectopic tissue formation. Data was analyzed sequentially. 11

12 The volume of the meniscus was determined for each of the postoperative time points using volume analysis postprocessing techniques. The volume of the meniscus was also compared with the postoperative volume of the meniscal remnant. This allowed for quantification of the degree of meniscal regeneration. The evaluation of MRI volume was challenging due to boundary detection and registration issues, so an additional over-read was performed. Also whole-organ evaluation of the knee in osteoarthritis (OA) was performed based on magnetic resonance imaging (MRI) findings, according to a validated method (Peterfy 2003). This is a semi-quantitative scoring method for overall evaluation of the osteoarthritic condition of the knee joint. This was performed by a contracted team of independent radiologists. Visual Analog Scale At the planned intervals, patients assessed their own perceptions of knee pain using a visual analog scale (VAS). The VAS was scored by measuring the distance of the mark along the scale from 0 (no pain) to 10 units (worst imaginable pain) and reporting a percentage value. Statistical Analysis Two-sided tests were used at a type I error rate of 5% for comparison between any 2 of the treatment groups or control and overall among the 3 treatment groups. Paired tests were used for significant changes from Baseline for any 1 of the 3 treatment groups. No adjustment for type I error was made for multiple treatment comparisons and testing multiple study endpoints. 12

13 Continuous data were described using descriptive statistics: n, mean, standard deviation, median, minimum, and maximum. For each study endpoint only observed data were used for analysis and no imputation of missing values was employed. All analyses were conducted using SAS software Version 8.2 (SAS Institute, Inc; Cary, NC) or higher. The completed population was defined as all randomly assigned patients who had the scheduled 6-month MRI evaluation, analyzed according to the treatment the patient actually received. The safety population was defined as all randomly assigned patients who received any study therapy, analyzed according to the treatment that the patient actually received. Categorical measures in ordinal scale were analyzed at each evaluation visit using the Mantel-Haenszel correlation test for pairwise treatment group comparisons and overall comparisons for the 3 treatment groups. Categorical measures in nominal scales were tested on general association between treatment group and outcomes at each visit using the Mantel-Haenszel test for general association. If any cell in a contingency table had a count less than 5, a Fisher exact test was used for nominal outcomes and exact P values of Mantel-Haenszel correlation tests were used for ordinal outcomes. Safety Analysis All safety data able to be verified against source documentation were used in safety analyses irrespective of the timing of adverse events (AEs), laboratory findings, or physical examinations. All AEs reported for this study were treatment emergent. Adverse events were coded according to Medical Dictionary for Regulatory Activities (MedDRA), Version

14 Adverse events were presented by system organ class and preferred term. The incidence of AEs table included only 1 occurrence of a preferred term per patient. If a patient reported the same preferred term multiple times, then that preferred term was counted only once, because patient counts (not the event counts) were presented. As with the preferred term, if a patient reported multiple AEs within the same system organ class, then that system organ class was counted only once. Percentages were calculated out of the number of patients in the safety analysis population. A summary of AEs by relationship to study drug was presented by incidence of occurrence. The event s relationship to study drug was classified as unrelated, possible, or probable. In the AE relationship table, if a patient reported multiple occurrences of the same AE, only the most closely related occurrence was presented. Results Basic demographics are presented in Table 1. The overall mean age for patients was 46.0 years with a minimum age of 18 years and maximum age of 60 years, consistent with the study eligibility criteria. Patients had a mean height of 174 cm, a mean weight of 87.0 kg, and a mean body mass index of 28.6 kg/m 2. No patients were unblinded prematurely during the study. Safety Evaluation No deaths were reported during the study and no AEs led to treatment discontinuation or study termination. A total of 427 AEs were experienced by the study subjects in the safety population, comprising patients who received any investigational agent. There were 118 in the vehicle control group, 158 in the Chondrogen 50 group, and 151 in the Chondrogen 150 group. Of the total number of AEs, 272 were reported as mild, 126 as 14

15 moderate, and 28 as severe. Adverse events that had an occurrence of at least 10% in any study group are summarized in Table 2. Nine SAEs occurred in 8 patients (Table 3), and all were determined by the blinded investigator to be unlikely related to investigational agent. Fifty-two of the 55 patients (94.5%) in the safety population reported at least 1 AE. The most frequently reported AEs by system organ class were musculoskeletal and connective tissue disorders (50/55, 90.9%), followed by general disorders and administration site conditions (27/55, 49.1%). Clinical laboratory testing There were no unexpected adverse trends identified in routine immunology, hematology, blood chemistry, and urinalysis analyses after study drug injection. No patients experienced a clinically significant abnormal hematology laboratory result. One patient experienced a clinically significant abnormal urinalysis laboratory result listed as crystals, however this patient had a history of nonclinically significant calcium oxalate crystals. The number of patients with shifts in laboratory values from normal at Baseline to values outside the normal range (low or high) or with shifts from normal to abnormal for categorical urinalysis analyses was generally low ( 4 patients per parameter). Vital signs, physical examination, and MRI safety. Two patients had vital sign measurements that were clinically significant and were reported as AEs. One patient experienced elevated blood pressure after injection of the study drug and 1 patient experienced an increase in body temperature. The 2 events were resolved. For physical examinations, there were no notable changes from Baseline in physical examination findings for all of the major organ system classes. There was no evidence of ectopic tissue formation from MRI evaluation. 15

16 MRI Evaluation The comparison of the Chondrogen groups versus the vehicle control group was analyzed for the percentage of patients with greater than 15% improvement in MRIbased meniscal volume from Baseline (post meniscectomy surgery and prior to investigation agent injection). The results of the analysis are shown in Table 4. At the 6 month evaluation, two patients (1 in each of the Chondrogen treatment groups) showed a change in meniscus volume greater than 15%. At 12 months, four patients in the Chondrogen 50 group showed a change in meniscus volume greater than 15%. Also, at Visit 7, three patients in the Chondrogen 50 treatment group showed a change in meniscus volume greater than 15%. At no time point was there the specific volume increase in any of the control group patients. At 12 months both the vehicle control versus Chondrogen 50 (P=0.040) and the overall group comparison (P=0.022) were statistically significant. At 2 years the overall group comparison (P=0.029) was statistically significant. The evaluation of MRI volume was challenging due to boundary detection and registration issues, so an additional second read was performed. The joint evaluation indicated 8 patients (44%) in the Chondrogen 50 group, 13 patients (72%) in the Chondrogen 150 group, and 9 patients (47%) in the Control group had degenerative changes consistent with OA. Bony changes associated with osteoarthritis, such as subchrondral sclerosis and osteophyte formation, were reported in 21% of patients receiving the control, but only 6% of Chondrogen-treated patients. 16

17 VAS Patients assessed perceptions of knee pain using a VAS. Overall, VAS scores decreased for patients post-surgery. The within-group P value revealed significance (P<0.001) in the reduction in pain when compared with baseline values as assessed by the VAS score for all treatment groups (Table 5). In patients with osteoarthritic-changes at the time of surgery, a statistically significant 20 mm reduction in pain, as measured by the visual analog scale (VAS), was observed in patients receiving a single injection of Chondrogen over patients receiving an injection of the control, HA, at one year (Chondrogen 150 : 47.6 mm vs. Chondrogen 50: 35.1 mm Control: 28.1 mm, p=0.05). The reduction in pain increased further with more severe osteoarthritic changes in the joint (p=0.004, Chondrogen 150: 55.7 mm vs. Chondrogen 50: 25.5 vs Control: 19.5 mm). These data are shown graphically in Figures 1 and 2. Discussion This was a Phase I/II, randomized, controlled double-blind study to evaluate the safety and preliminary efficacy of hmscs delivered by intra-articular injection after partial meniscectomy. In the study 55 male and female patients who required medial meniscectomy and were otherwise healthy were treated. Patients were followed for 2 years from the meniscectomy surgery. Overall, the study injections were well tolerated with minimal adverse effects. There were no deaths or AEs that led to treatment discontinuation or study termination. The most frequently reported AEs by preferred term were arthralgia, joint swelling, joint stiffness, injection site joint pain, joint effusion, headache, and peripheral edema. Many of the listed AEs such as injection site joint pain, joint swelling/effusion, and headache 17

18 were commonly reported with the use of sodium hyaluronan, the vehicle control used in this study. Eight patients reported SAEs, and all were considered by the investigator as unlikely to be related to study drug. Sequential immunologic, hematologic, and urine testing showing no unexpected adverse trends. A few patients experienced clinically significant abnormal laboratory results; however, none of these were considered to be related to study drug. In addition, there was no evidence of ectopic tissue formation in the knee joint. This study demonstrated that MSCs can be delivered in a concentrated manner to an enclosed space, here the joint capsule, with signs of abnormal tissue formation. This finding is consistent with other results of systemic administration of MSCs that have shown no evidence of unwanted tissue formation from imaging studies of patients receiving these cells. (Hare 2009, Kebriaei 2009). The results of this study suggest that MSCs have to potential to improve the overall joint condition. This study investigated only a single treatment of stem cells; multiple or serial stem cell injection protocols might bring greater clinical changes. For some patients receiving MSCs, there was an increase in meniscal volume indicating de novo tissue regeneration. Perhaps more importantly for the patients, a higher proportion of those with osteoarthritic changes experienced a reduction in pain. The reduction in pain was relative to an active control, hyaluronic acid. In fact, hyaluronic acid is often a treatment for the pain of osteoarthritis itself and possibly was providing some benefit to the patients in the control group. The magnitude of effect relative to placebo was clinically meaningful. The observed effect on a patient-reported outcome was 18

19 compelling given that this was a double blind study, which eliminates the possible bias from knowing that one received stem cells. The ability to modify the course of OA, especially in patients following removal of the meniscus, which serves to protect the joint, is a tantalizing possibility that requires further investigation. Currently no available therapies have been proven to be disease modifying. Standard therapy for OA of the knee begins with over-the-counter pharmacologic agents such as acetaminophen and nonsteroidal anti-inflammatory drugs. Treatment then progresses to agents including codeine and propoxyphene and COX-2 specific inhibitors. The last line of therapy before a final outcome of total knee replacement is often intra-articular injection of corticosteroid or hyaluronic acid into the knee joint. Difficulties with consistency of MRI scans from different centers, visit to visit, and edge detection were some of the challenges encountered with attempting quantitative MRI analysis of the knee. Further work is needed to validate quantitative assessment of meniscal volumes. While evaluation of meniscal volumes was initiated in an effort to demonstrate the mechanism of MSCs in knee, further studies would need to be designed around and focus on the patient reported outcomes, which are required for products/drugs under development in this area. Conclusion While further research will further elucidate the effects of MSC administration on the condition of the knee joint, this study provided the first controlled human data on allogeneic MSCs. In terms of safety there were no adverse effects from these cells. 19

20 Whether providing additional treatments (i.e. injections) enhances the effect on pain or regeneration remains to be evaluated. Many joint treatments are given repeatedly and MSCs multiply dosed might take advantage of the anti-inflammatory effects of MSC administration. 20

21 Acknowledgments This study was sponsored by Osiris Therapeutics, Inc. (Baltimore, MD). The investigators would like to thank the people who agreed to participate in this trial and the study contributors, especially Dr. Wendy Burke. 21

22 Table 1: Patient Disposition and Demographics Vehicle Control (n=20) Chondrogen 50 (n=20) Chondrogen 150 (n=20) Total (N=60) Number of Patients Randomized 20 (100.0%) 20 (100.0%) 20 (100.0%) 60 (100.0%) Completed 17 (85.0%) 17 (85.0%) 18 (90.0%) 52 (86.7%) Discontinued 3 (15.0%) 3 (15.0%) 2 (10.0%) 8 (13.3%) Completed Population 19 (95.0%) 17 (85.0%) 18 (90.0%) 54 (90.0%) Age (years) N Mean (SD) 47.8 (8.00) 44.6 (9.82) 45.6 (12.42) 46.0 (10.16) Median Min, Max 24, 60 24, 57 18, 60 18, 60 Sex - n (%) Male 13 (65.0) 11 (55.0) 14 (70.0) 38 (63.3) Height (cm) N Mean (SD) (11.894) (9.021) (11.190) (10.734) Median Min, Max 153.2, , , , Weight (kg) N Mean (SD) (17.292) (21.422) (22.614) (20.331) Median Min, Max 49.5, , , , Body-Mass Index (kg/m 2 ) N Mean (SD) (4.049) (7.943) (5.909) (6.201) Median Min, Max 19.1, , , , 45.5 Abbreviations: Max = maximum; Min = minimum. Note: Percentages are based on the number of patients in each treatment group. 22

23 Table 2: Adverse Events Occurring in 10% of Patients System Organ Class Preferred Term Control (n=19) Chondrogen 50 (n=18) Chondrogen 150 (n=18) Total number of adverse events Number of patients with at least 1 AE 17 (89.5%) 18 (100.0%) 17 (94.4%) Gastrointestinal disorders 2 (10.5%) 2 (11.1%) 1 (5.6%) Nausea 0 2 (11.1%) 1 (5.6%) General disorders/administration site conditions 6 (31.6%) 10 (55.6%) 11 (61.1%) Injection site joint pain 5 (26.3%) 3 (16.7%) 6 (33.3%) Injection site joint swelling 0 2 (11.1%) 3 (16.7%) Injection site pain 0 2 (11.1%) 2 (11.1%) Oedema peripheral 2 (10.5%) 4 (22.2%) 3 (16.7%) Infections and infestations 3 (15.8%) 3 (16.7%) 3 (16.7%) Nasopharyngitis 1 (5.3%) 0 2 (11.1%) Sinusitis 1 (5.3%) 1 (5.6%) 2 (11.1%) Injury, poisoning, procedural complications 6 (31.6%) 8 (44.4%) 8 (44.4%) Contusion 2 (10.5%) 0 2 (11.1%) Excoriation 1 (5.3%) 2 (11.1%) 0 Meniscus lesion 1 (5.3%) 2 (11.1%) 2 (11.1%) Muscle strain 1 (5.3%) 0 2 (11.1%) Joint sprain 1 (5.3%) 2 (11.1%) 2 (11.1%) Procedural pain 1 (5.3%) 0 3 (16.7%) Investigations 3 (15.8%) 6 (33.3%) 2 (11.1%) Blood triglycerides increased 1 (5.3%) 2 (11.1%) 1 (5.6%) Musculoskeletal and connective tissue disorders 15 (78.9%) 18 (100.0%) 17 (94.4%) Arthralgia 13 (68.4%) 17 (94.4%) 15 (83.3%) Back pain 0 2 (11.1%) 1 (5.6%) Joint crepitation 0 1 (5.6%) 3 (16.7%) Joint effusion 3 (15.8%) 4 (22.2%) 2 (11.1%) Joint range of motion decreased (27.8%) Joint stiffness 4 (21.1%) 7 (38.9%) 5 (27.8%) Joint swelling 8 (42.1%) 8 (44.4%) 10 (55.6%) Muscle atrophy 2 (10.5%) 1 (5.6%) 0 Muscular weakness 0 2 (11.1%) 2 (11.1%) Musculoskeletal pain 1 (5.3%) 1 (5.6%) 2 (11.1%) Myalgia 0 2 (11.1%) 0 Osteoarthritis 1 (5.3%) 2 (11.1%) 3 (16.7%) Pain in extremity 1 (5.3%) 1 (5.6%) 3 (16.7%) Rotator Cuff Syndrome 2 (10.5%) 0 0 Synovial Cyst 0 3 (16.7%) 1 (5.6%) Tendonitis 2 (10.5%) 2 (11.1%) 0 23

24 24

25 Table 3: Serious Adverse Events Event Control (n=19) Chondrogen 50 x 10 6 cells (n=18) Chondrogen 150 x 10 6 cells (n=18) Acute myocardial infarction Ileus Small intestinal obstruction Femur fracture Fibula fracture Hand fracture Meniscus lesion Osteoarthritis

26 Table 4: Magnetic Resonance Imaging Results for Meniscal Volume Control (n=19) Chondrogen 50 (n=17) Chondrogen 150 (n=18) >15% Change in Meniscus Volume Relative to Baseline 6 months n (%) 0 1 (5.9%) 1 (5.6%) 95% CI for Percentage (0.0, 17.6) (0.1, 28.7) (0.1, 27.3) P Value* for Vehicle Versus Vehicle Versus Versus 50 >0.999 Overall months n (%) 0 4 (23.5) 1 (5.6) 95% CI for Percentage (0.0, 17.6) (6.8, 49.9) (0.1, 27.3) P Value* for Vehicle Versus Vehicle Versus Versus Overall years n (%) 0 3 (17.6) 0 95% CI for Percentage (0.0, 19.5) (4.0, 45.6) (0.0, 19.5) P Value* for Vehicle Versus 150 >0.999 Vehicle Versus Versus Overall Abbreviations: CI = confidence interval. * P values are for the 3 pairwise treatment group actual result comparisons and the overall 3 treatment group comparison using the Mantel-Haenszel general association test. 26

27 Table 5: Absolute improvement in VAS relative to baseline Overall n 6 weeks 3 months 6 months 1 year Chondrogen Chondrogen Control OA n 6 weeks 3 months 6 months 1 year Chondrogen Chondrogen Control More severe OA 6 weeks 3 months 6 months 1 year Chondrogen Chondrogen Control

28 Figure 1: Improvement in Pain Score relative to vehicle control in the presence of osteoarthritic changes based on MRI. 28

29 Figure 2: Improvement in Pain Score relative to vehicle control in the presence of moderate to severe osteoarthritic changes based on MRI. 29