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An Initial Injection and a Crossover Injection of Amniotic Suspension Allograft Following Failed Treatment with Hyaluronic Acid or Saline Are Equally Effective in the Treatment of Moderate Symptomatic Knee Osteoarthritis Over 12 Months
The purpose of this crossover study was to determine the efficacy of amniotic suspension allograft (ASA) for moderate symptomatic knee osteoarthritis following failed treatment with hyaluronic acid (HA) or saline through 12 months’ postcrossover injection using patient-reported and safety outcomes.
Methods
In this multicenter study, 95 patients from a 200-patient single-blind randomized controlled trial were eligible to crossover and receive a single injection of ASA 3 months after failed treatment with HA or saline. Patient-reported outcomes, including Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS), were collected out to 12 months postcrossover to determine pain and function. Radiographs and blood were collected for assessment of changes. Statistical analyses were performed using mixed effects model for repeated measures.
Results
Treatment with ASA following failed treatment with HA or saline resulted in significant improvements in KOOS and VAS scores compared with crossover baseline. There were no differences in radiographic measures or anti-human leukocyte antigen serum levels compared with baseline and no severe adverse events reported. In addition, more than 55% of patients were responders at months 3, 6, and 12 as measured by the Outcome Measures in Arthritis Clinical Trials—Osteoarthritis Research Society International simplified responder criteria. There were no significant differences between the original ASA randomized group and crossover cohorts at any of the time points evaluated, suggesting that prior failed treatment with HA or saline did not significantly impact outcomes following treatment with ASA.
Conclusions
This study showed that patients who previously failed treatment with HA or saline had statistically significant improvements in pain and function scores following a crossover injection of ASA that was sustained for 12 months, as measured by KOOS and VAS. There were no serious adverse events reported, and the injection was safe.
Level of Evidence
II, prospective cohort study.
Placental-derived tissues were originally used in the early 1900s to treat nonhealing wounds, ulcers, and burns.
to identify whether these allografts could be a potential nonsurgical treatment for symptomatic knee osteoarthritis (OA). Mechanistically, several anti-inflammatory proteins and protease inhibitors have been identified in placental-derived tissues
; we hypothesize these factors may help reduce the inflammation perpetuated by proinflammatory cytokines including interleukin-1 and tumor necrosis factor-α present in the OA microenvironment.
using the rat medial meniscal transection model to induce OA showed improved proteoglycan concentrations and lower numbers of cartilage lesions/erosions following treatment with an injectable dehydrated amnion/chorion membrane. A second rat study using the medial meniscal transection model with an injectable particulated amniotic membrane/umbilical cord matrix showed lower cartilage degeneration and increased cartilage volume and thickness.
Another rat study using the monosodium iodoacetate model for chemically inducing OA showed improvements in pain and function as measured by behavioral testing, along with increases in interleukin-10 levels in the synovial fluid following treatment with amniotic suspension allograft (ASA).
A chemically induced rabbit OA model using collagenase type II showed a delay in histologic changes of cartilage in the knee following treatment with an injectable lyophilized amniotic membrane.
ASA contains micronized human amniotic membrane and cells from the amniotic fluid and is cryopreserved. These cells and tissues are obtained from consenting human donors during elective cesarean deliveries following all applicable standards of the Food and Drug Administration and the American Association of Tissue Banks.
ASA was first studied in a 6-patient open-label single-arm study due to its potential to modulate inflammation; this study supported the safety of a single injection of ASA over 12 months.
While trends were seen with patient-reported outcomes (PROs), the study was not powered to demonstrate treatment efficacy. The follow-up study, a 200-patient single-blind, randomized controlled trial at 12 sites across the United States, compared a single injection of ASA to both hyaluronic acid (HA, standard of care) and saline (placebo control). In this study, ASA was shown to result in statistically significant improvements in pain and function scores compared with saline and HA for up to 12 months.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
In the randomized trial, subjects self-reporting unacceptable pain at 3 months were eligible to participate in a crossover arm. Crossover study designs are commonly used in trials to allow more trial subjects access to the investigational treatment after having failed at achieving one or more pre-defined study endpoints.
Since many subjects were treated with saline, the study design and inclusion of a crossover arm was implemented to improve enrollment and as an ethical measure to ensure that patients who did not find pain relief had the option to be treated with the investigational study product. The purpose of this crossover study was to determine the efficacy of ASA for moderate symptomatic knee OA following failed treatment with HA or saline through 12 months postcrossover injection using patient-reported outcomes and safety outcomes. The hypothesis of this study was that there would be improvements in PROs from crossover baseline up to 12 months, and no safety concerns following an injection of ASA.
Methods
This manuscript details the results from the ASA crossover arm, which was part of a prospective, multi-center, single-blinded, randomized controlled trial (ClinicalTrials.gov: NCT02318511) that initially enrolled 200 adult patients with symptomatic knee OA at 12 study sites across the United States that met defined inclusion/exclusion criteria.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
Of the 200 patients originally enrolled, 104 patients self-reported unacceptable pain at 3 months, with 44 and 51 of those from the HA and saline groups, respectively. All patients who were eligible (n = 95) elected to participate in the prospective, unblinded ASA crossover arm. Because the original study called for a single injection at baseline, all subjects in the crossover arm had a 3-month period of treatment with HA, or a 3-month period with saline before receiving ASA treatment in the crossover arm (baseline to 3 months in the original study).
Patients were enrolled in the initial prospective study under a Western Institutional Review Board (#20142125)-approved protocol after signing an informed consent protocol between June 2015 and July 2017. Inclusion criteria included adult patients aged 18 years and older with a body mass index (BMI) less than 40, a confirmed diagnosis of moderate knee OA through a Kellgren–Lawrence grade of 2 or 3,
and a 7-day average pain score of 4 or greater on a scale of 1-10. If female, they were abstinent, surgically sterile, actively practicing an accepted contraceptive method, or postmenopausal.
Exclusion criteria included use of pain medications less than 15 days before the injection, use of pain medication other than acetaminophen for conditions unrelated to OA of the index knee, regular use of anticoagulants, patients with a history of substance abuse, or patients who failed to agree not to take additional knee symptom modifying drugs during the course of the study without reporting the medication use to the study team. Knee-related treatment exclusion criteria included knee surgery on the index knee within 12 months or on the contralateral knee within 6 months, acute injury to the index knee within 3 months, intra-articular injections with either corticosteroids or viscosupplementation in the index knee within 3 months, or confirmed mechanical symptoms such as locking, intermittent block to range of motion, or loose body sensations often associated with intra-articular loose bodies or meniscal displacements. Additional exclusion criteria included current immunosuppressive treatment (including corticosteroids at a dose greater than 5 mg/day), rheumatoid arthritis and other autoimmune disorders, history of diabetes mellitus, infection requiring antibiotic treatment within 3 months, diagnosis of malignancy, apart from treated basal cell cancer of the skin, within the last 5 years, history of solid-organ or hematologic transplantation, or workers’ compensation patients. Female patients were excluded if they had a desire to become pregnant during the course of the study or were pregnant.
Patients were randomized 1:1:1 to either saline, HA, or ASA and blinded to their injection.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
Injections were as follows: ASA (2.0 mL ReNu diluted with 2.0 mL injectable sterile normal saline; Organogenesis, Canton, MA); HA (4.0 mL Monovisc High Molecular Weight Hyaluronan; Anika Therapeutics, Boston, MA); injectable sterile normal saline (4.0 mL). HA was chosen as it was the standard of care injectable at the time of the study initiation. At 3 months, patients who self-reported unacceptable pain were eligible to participate in the ASA crossover arm if their original randomization was saline or HA. If they were originally randomized to the ASA group, patients were considered a treatment failure and their care was managed outside the study with standard of care. The Consolidated Standards of Reporting Trials diagram illustrating the enrollment, allocation, and disposition of patients in the crossover study is shown in Figure 1. A previous power analysis was completed as described,
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
resulting in a sample size of 32 patients per group; the final sample size number was 61 per group to account for study dropouts.
Fig 1Consolidated Standards of Reporting Trials (CONSORT) flow diagram used to describe the grouping and flow of patients through the clinical trial. (ASA, amniotic suspension allograft; HA, hyaluronic acid.)
As a part of the crossover arm, all patients had a new crossover baseline evaluation that included a physical examination, medical and knee history, radiographs, and blood draws for laboratory analyses. Questionnaires were collected by research assistants or study staff during in-person follow-up visits, while the physical examination and radiographic assessments were performed by the site principal investigators. Patients completed the following PROs: EuroQol 5 Dimension 5 Level (global health), Knee Injury and Osteoarthritis Outcome Score (KOOS), Single Assessment Numerical Evaluation (SANE), visual analog scale (VAS) using a 150-mm scale, and the Tegner Activity Scale. The KOOS subscores examined included KOOS Pain, KOOS Symptoms, KOOS Activities of Daily Living (ADL), KOOS Sports & Recreation, and KOOS Quality of Life (QoL). The VAS questionnaire included questions of VAS Overall Pain, VAS Strenuous Work Pain, VAS Sedentary Work Pain, and VAS Normal Daily Living (NDL) Pain. Minimal important difference (MID) also was evaluated; the MID has been established as 12 points for KOOS Pain with a range of 4 to 20, 8 points for KOOS ADL with a range of 3 to 9,
Application of minimal important differences in degenerative knee disease outcomes: A systematic review and case study to inform BMJ Rapid Recommendations.
Due to the nature of the study, there was no blind in place for subjects, investigators, or study staff during the crossover period. Subjects and investigators in the crossover arm were unblinded due to study design and description in the informed consent that if they received a second injection, it would be ASA; these subjects received a single intra-articular injection of ASA. Subsequent follow-up visits occurred at 1 week, 6 weeks, 3 months, 6 months, and 12 months postcrossover, resulting in 15 months of total study participation from the primary baseline injection. Additional blood draws were obtained at 6 week and 6 months following the crossover baseline evaluation, and final radiographs were captured at 12 months. Blood analyses included examination of complete blood count, basic metabolic profile, C-reactive protein (CRP), immunoglobulin levels (including immunoglobulin A, immunoglobulin E, immunoglobulin G, and immunoglobulin M), and anti-human leukocyte antigen (anti-HLA) responses. Anti-HLA testing was performed to determine whether ASA injections modified the presence of class I anti-HLA antibodies in the patients’ serum. For anti-HLA analyses, methods are detailed elsewhere.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
To summarize, patients’ serum samples were examined for the presence of anti-HLA antibodies using the One Lambda (West Hills, CA) LABScreen Mixed Class I & II (LSM12). Any positive or undefined samples were then further tested for Class I anti-HLA antibodies using the LABScreen Single Antigen HLA Class I assay (LS1A04). Changes in CRP, immunoglobulins, anti-HLA, and incidence of treatment emergent adverse events (TEAEs) were assessed as safety outcomes. Radiographic measurements examined included affected knee, worst affected compartment, Kellgren-Lawrence grade, and joint space narrowing.
Because the crossover population was recruited from 2 of the 3 original groups, HA and saline, these groups were directly compared across the entire 15-month study timeline, from the original baseline to the 3-month visit (blinded) and from the crossover baseline to the end of the study at the 12-month time point (unblinded) to determine comparability of responses between the groups. Combined HA and saline groups (n = 95) change from baseline to 3 months was then compared between the original period and the crossover period. All data analyses and statistics were performed by a statistician independent of the study. For missing data, the mixed effect model for repeated measures was used as a sensitivity analysis. SAS PROC MIXED was used to determine P values for the original randomized cohort versus the crossover cohort, where the baseline values for each measure was included as a covariate and fixed factors included treatment and visit. Interaction terms in this model included baseline-by-visit and treatment-by-visit. The repeated factor was the visit within subject, and an unstructured covariance was used. P values for categorical variables were from chi-squared (demographics, radiographic data), while P values for quantitative variables were from generalized linear model (demographics, radiographic data). For the combined crossover cohort, within group changes from baseline were determined by a paired t-test. For all P values, P < .05 was considered statistically significant. For the Outcome Measures in Arthritis Clinical Trials—Osteoarthritis Research Society International (OMERACT-OARSI) responder analysis, data imputation using Last observation carried forward was analyzed for responders if they met the requirement for high improvement (≥50% improvement in pain, function, or quality of life and an absolute change of ≥20 points) or improvement (≥20% improvement in 2 of the following—pain, function, or quality of life—and an absolute change of ≥10 points).
P values for the OMERACT-OARSI responder rate between the HA and saline groups were from PROC FREQ. Conversion of the KOOS scores to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was done by taking components of the KOOS subscores and summing them. WOMAC pain consists of KOOS Pain items P5-P9; WOMAC stiffness consists of KOOS Symptoms items S6-S7, and WOMAC function consists of KOOS ADL items A1-A17, as described.
Patient Baseline Demographics and Radiographic Values
In total, 95 patients who reported unacceptable pain from the initial prospective study participated in the ASA crossover arm, and treatment efficacy of a single injection of ASA over 12 months was assessed using PROs. There were no statistically significant differences in baseline demographics (Table 1) and radiographic data (Table 2) between the HA and saline groups.
Table 1Crossover Baseline Demographic Values
HA (n = 44)
Saline (n = 51)
Statistics
Age, y
55.7 ± 1.6
54.1 ± 1.4
P = .4336
Sex
P = .7287
Female
20 (45.5%)
23 (45.1%)
Male
24 (54.5%)
28 (54.9%)
Height, inches
68.4 ± 0.6
68.3 ± 0.5
P = .8494
Weight, pounds
188.1 ± 6.5
189.8 ± 5.1
P = .8357
BMI
28.0 ± 0.7
28.5 ± 0.6
P = .5645
NOTE. Mean ± standard error of the mean reported when applicable; otherwise, number of patients for each category reported along with percentage of total patients. P values for categorical variables from χ2 test; P values for quantitative variables from GLM.
BMI, body mass index; GLM, generalized linear model; HA, hyaluronic acid.
NOTE. Number of patients for each category reported and percentage of total patients reported for each treatment group; mean ± standard error of the mean reported for joint space narrowing. P values for categorical variables (affected knee, worst compartment, KL grade) from χ2 test; P values for quantitative variables (joint space narrowing) from GLM.
GLM, generalized linear model; HA, hyaluronic acid; KL, Kellgren-Lawrence; mm, millimeters.
Comparison of Crossover Cohort by Original Randomization Group
Three subscores (KOOS Pain, KOOS ADL, and VAS Overall Pain) are shown in Figure 2. For the KOOS pain subscale, there were no significant differences between the HA and saline group except at the 1-week visit following their initial randomization, where the HA group was higher (Fig 2A, P = .0299). KOOS ADL and VAS Overall Pain is shown in Figure 2 B and C; neither of these outcome measures showed significant differences between the HA and saline groups throughout the entire 15-month study period. There were no statistically significant differences in the OMERACT-OARSI responder rates and the radiographic outcomes between the HA and saline groups. Because of the comparability between the groups, the entire crossover cohort was combined for further analyses; raw scores for the PROs assessed for the HA and saline cohorts separately can be found in Table 3.
Fig 2Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS) scores for the entire course of treatment, including original randomization and crossover period plotted as a function of the patients’ original randomization group. Mean ± standard error of the mean reported for (A) KOOS Pain, (B) KOOS Activities of Daily Living, and (C) VAS Overall Pain. P values from PROC MIXED. ∗Denotes P < .05. (HA, hyaluronic acid; mo, month; wk, week.)
Table 3Patient-Reported Outcomes for Crossover Cohorts Separated by Original Randomization Cohort.
HA (n = 44)
Saline (n = 51)
Crossover Baseline
1 Week
6 Weeks
3 Months
6 Months
12 Months
Crossover Baseline
1 Week
6 Weeks
3 Months
6 Months
12 Months
EQ-5D-5L
Mobility
2.2 ± 0.1
2.0 ± 0.1
1.8 ± 0.1
1.7 ± 0.1
1.8 ± 0.2
1.6 ± 0.1
2.2 ± 0.1
2.0 ± 0.1
1.8 ± 0.1
1.7 ± 0.1
1.4 ± 0.1
1.6 ± 0.1
Self-Care
1.3 ± 0.1
1.3 ± 0.1
1.1 ± 0.1
1.2 ± 0.1
1.2 ± 0.1
1.1 ± 0.1
1.4 ± 0.1
1.3 ± 0.1
1.2 ± 0.1
1.2 ± 0.1
1.1 ± 0.0
1.0 ± 0.0
Activities
2.3 ± 0.1
2.0 ± 0.1
1.7 ± 0.1
1.6 ± 0.1
1.8 ± 0.2
1.5 ± 0.1
2.2 ± 0.1
2.0 ± 0.1
1.7 ± 0.1
1.7 ± 0.1
1.4 ± 0.1
1.7 ± 0.1
Pain
2.7 ± 0.1
2.3 ± 0.1
2.2 ± 0.1
2.1 ± 0.1
2.3 ± 0.1
2.0 ± 0.2
2.8 ± 0.1
2.4 ± 0.1
2.2 ± 0.1
2.3 ± 0.1
2.1 ± 0.1
2.1 ± 0.1
Anxiety
1.5 ± 0.1
1.4 ± 0.1
1.4 ± 0.1
1.4 ± 0.1
1.4 ± 0.1
1.2 ± 0.1
1.5 ± 0.1
1.4 ± 0.1
1.4 ± 0.1
1.3 ± 0.1
1.2 ± 0.1
1.3 ± 0.1
Health Today
81.5 ± 1.9
83.8 ± 1.7
87.1 ± 1.6
87.6 ± 1.4
86.4 ± 2.0
87.6 ± 2.2
76.1 ± 2.0
78.7 ± 1.9
83.2 ± 1.8
82.6 ± 2.1
85.6 ± 1.7
85.4 ± 2.3
KOOS
Pain
59.0 ± 2.7
64.0 ± 2.9
69.2 ± 3.0
69.5 ± 3.7
67.5 ± 4.2
70.4 ± 4.5
56.5 ± 2.6
61.6 ± 2.4
68.1 ± 2.7
68.0 ± 2.5
70.2 ± 3.1
67.1 ± 3.5
Symptoms
50.7 ± 2.2
56.4 ± 2.3
58.2 ± 2.5
55.5 ± 2.8
54.9 ± 3.1
57.9 ± 3.5
47.8 ± 1.8
48.7 ± 2.3
53.4 ± 2.1
52.9 ± 2.2
54.1 ± 2.4
54.6 ± 2.8
Activities of Daily Living
66.8 ± 3.1
72.3 ± 3.0
76.8 ± 3.0
76.4 ± 4.0
73.9 ± 4.4
77.6 ± 4.5
66.0 ± 2.6
69.1 ± 2.5
72.7 ± 3.1
75.4 ± 2.3
77.1 ± 3.1
75.4 ± 3.6
Sports & Recreation
38.7 ± 3.3
42.1 ± 3.8
50.7 ± 3.6
49.7 ± 4.3
50.3 ± 5.0
52.8 ± 4.8
35.6 ± 2.9
42.4 ± 3.1
50.0 ± 3.7
51.8 ± 3.2
53.0 ± 3.6
51.7 ± 4.3
Quality of Life
40.4 ± 3.0
44.9 ± 3.2
50.8 ± 3.4
50.4 ± 4.0
51.6 ± 4.4
51.9 ± 4.8
34.5 ± 2.6
39.2 ± 2.4
45.1 ± 3.0
46.5 ± 2.8
51.4 ± 3.3
44.2 ± 4.4
SANE
61.0 ± 2.9
71.0 ± 3.4
78.7 ± 3.0
81.6 ± 2.6
77.2 ± 3.7
81.3 ± 3.4
58.4 ± 3.2
68.7 ± 2.6
72.3 ± 2.9
72.7 ± 3.0
77.2 ± 3.0
71.5 ± 3.6
Tegner
3.9 ± 0.3
3.8 ± 0.3
4.2 ± 0.3
4.6 ± 0.3
4.1 ± 0.4
4.4 ± 0.3
3.5 ± 0.3
3.7 ± 0.3
4.3 ± 0.3
3.9 ± 0.3
4.4 ± 0.3
4.6 ± 0.3
VAS
Overall Pain
67.5 ± 5.4
47.3 ± 5.6
36.1 ± 4.4
34.8 ± 5.2
38.8 ± 6.4
31.8 ± 5.8
70.5 ± 4.8
56.7 ± 4.7
48.4 ± 5.1
46.8 ± 5.1
42.5 ± 5.4
43.5 ± 6.4
Strenuous Work Pain
89.1 ± 5.4
70.3 ± 6.2
60.8 ± 6.0
53.5 ± 7.0
57.5 ± 7.3
49.2 ± 7.2
88.3 ± 5.1
77.2 ± 5.0
67.6 ± 5.5
68.4 ± 5.6
58.6 ± 5.9
62.7 ± 7.2
Sedentary Work Pain
34.8 ± 4.5
28.0 ± 4.9
19.5 ± 3.9
18.1 ± 4.1
21.8 ± 4.5
14.2 ± 4.7
42.2 ± 4.9
36.2 ± 4.7
31.3 ± 4.5
28.5 ± 4.3
19.9 ± 3.5
20.0 ± 4.1
Normal Daily Living Pain
57.7 ± 5.7
45.3 ± 5.5
33.3 ± 4.8
29.3 ± 5.6
35.3 ± 6.3
25.3 ± 5.2
60.0 ± 5.0
53.2 ± 5.1
46.3 ± 5.2
38.5 ± 4.5
36.8 ± 5.4
36.5 ± 5.9
Responder rate
OMERACT-OARSI Simplified
N/A
N/A
N/A
27 (61.4%) p=0.4085
27 (61.4%) p=0.8899
27 (61.4%) p=0.3096
N/A
N/A
N/A
27 (52.9%)
32 (62.7%)
26 (51.0%)
High Improvement
N/A
N/A
N/A
19 (43.2%) p=0.0689
18 (40.9%) p=0.7157
19 (43.2%) p=0.4318
N/A
N/A
N/A
13 (25.5%)
19 (37.3%)
18 (35.3%)
Improvement
N/A
N/A
N/A
24 (54.5%) p=0.7286
24 (54.5%) p=0.6746
24 (54.5%) p=0.2672
N/A
N/A
N/A
26 (51.0%)
30 (58.8%)
22 (43.1%)
NOTE. Mean ± standard error of the mean reported for each treatment group.
EQ-5D-5L, EuroQol 5 Dimension 5 Level; HA, hyaluronic acid, KOOS, Knee Injury and Osteoarthritis Outcome Score; N/A, not available; OMERACT-OARSI, Outcome Measures in Arthritis Clinical Trials—Osteoarthritis Research Society International; SANE, Single Assessment Numerical Evaluation, VAS, visual analog scale. P values for responder rates from PROC FREQ between HA and saline groups.
Three-Month Comparison of Original Randomization Treatment to ASA Injection for Crossover Cohort
When comparing changes from baseline to the 3 month visit for the original randomization period and changes from crossover baseline to the 3 month visit for the crossover period (Fig 3), we found there was a significant difference in changes from baseline scores in the KOOS Pain (P = .0044), KOOS ADL (P = .0432), VAS Overall Pain (P = .0064), VAS Sedentary Work Pain (P = .0244), and the VAS NDL Pain (P = .0059).
Fig 3Change from baseline for Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS) scores for the first 3 months of the original treatment period and first 3 months of the crossover period for the combined crossover cohort (n = 95). Mean ± standard error of the mean reported for (A) KOOS and (B) VAS. P values from PROC MIXED. ∗Denotes P < .05; ˆdenotes P < .01.
None of the reported MIDs were reached for the crossover cohort’s initial 3-month period (white bars, Fig 3). However, during the crossover period, average improvements that met or exceeded the MID were KOOS Pain, KOOS ADL, and VAS Overall Pain (Fig 3).
PROs for Crossover Cohort Following ASA Injection
For the 12-month follow-up period, there were significant improvements from crossover baseline at all time points, with scores improving from 57.7 ± 1.9 (average ± standard error of the mean) to 68.6 ± 2.8 at 12 months for KOOS Pain (P = .0003; Fig 4A). KOOS Symptoms (Fig 4B) significantly improved from 49.2 ± 1.4 to 56.1 ± 2.2 at 12 months (P = .0020). KOOS ADL improved from 66.4 ± 2.0 to 76.4 ± 2.8 at 12 months (P = .0015; Fig 4C), whereas KOOS Sports and Recreation and KOOS QoL also improved from 37.0 ± 2.2 to 52.2 ± 3.2 at 12 months and from 37.2 ± 2.0 to 47.6 ± 3.3 at 12 months, respectively (P < .0001 and P = .0005; Fig 4 D and E). Based on the change from baseline values, the MID was attained or exceeded for the KOOS Pain and ADL subscores at 12 months.
Fig 4Knee Injury and Osteoarthritis Outcome Score (KOOS) for the combined crossover cohort (n = 95). Mean ± standard error of the mean reported for (A) KOOS Pain, (B) KOOS Symptoms, (C) KOOS Activities of Daily Living, (D) KOOS Sports & Recreation, and (E) KOOS Quality of Life. P values are for within-group changes from baseline P values using the paired t-test. ∗Denotes P < .05; ˆdenotes P < .01, #denotes P < .001, and ‡denotes P < .0001. (ASA, amniotic suspension allograft; mo, month; wk, week.)
VAS scores for the combined cohort were significantly improved at all visits compared with the crossover baseline (Fig 5). VAS Overall Pain improved from 69.1 ± 3.5 to 38.4 ± 4.4 (44.4% improvement; P < .0001) at 12 months, whereas VAS Strenuous Work Pain improved from 88.6 ± 3.7 to 56.8 ± 5.2 at 12 months (36.0%; P < .0001). Similarly, VAS Sedentary Work Pain decreased from 38.8 ± 3.3 to 17.4 ± 3.1 at 12 months (55.1%; P < .0001), while VAS NDL pain decreased from 58.9 ± 3.7 to 31.6 ± 4.1 at 12 months (46.4%; P < .0001). Based on the change from baseline values, the MID was attained or exceeded for VAS Overall Pain. Additional PRO values assessed including EuroQol 5 Dimension 5 Level, Single Assessment Numerical Evaluation, and Tegner Activity Scale are listed in Table 4.
Fig 5Visual analog scale (VAS) Scores for the combined crossover cohort (n = 95). Mean ± standard error of the mean reported for (A) VAS Overall Pain, (B) VAS Strenuous Work Pain, (C) VAS Sedentary Work Pain, and (D) VAS Normal Daily Living Pain. P values are for within-group changes from baseline p-values using the paired t-test. ∗Denotes P < .05; ˆdenotes P < .01, and ‡denotes P < .0001. (ASA, amniotic suspension allograft; mo, month; wk, week.)
Table 4Patient-Reported Outcomes For Combined Crossover Cohort
Crossover Baseline
1 Week
6 Weeks
3 Months
6 Months
12 Months
EQ-5D-5L
Mobility
2.2 ± 0.1
2.0 ± 0.1 P = .0135
1.8 ± 0.1 P < .0001
1.7 ± 0.1 P < .0001
1.6 ± 0.1 P < .0001
1.6 ± 0.1 P < .0001
Self-Care
1.3 ± 0.1
1.3 ± 0.1 P = .4357
1.2 ± 0.1 P = .0034
1.2 ± 0.0 P = .0060
1.1 ± 0.0 P = .0275
1.1 ± 0.0 P = .0002
Activities
2.3 ± 0.1
2.0 ± 0.1 P = .0064
1.7 ± 0.1 P < .0001
1.6 ± 0.1 P < .0001
1.6 ± 0.1 P < .0001
1.6 ± 0.1 P < .0001
Pain
2.8 ± 0.1
2.4 ± 0.1 P < .0001
2.2 ± 0.1 P < .0001
2.2 ± 0.1 P < .0001
2.2 ± 0.1 P < .0001
2.1 ± 0.1 P < .0001
Anxiety
1.5 ± 0.1
1.4 ± 0.1 P = .0948
1.4 ± 0.1 P = .2768
1.4 ± 0.1 P = .3569
1.3 ± 0.1 P = .2700
1.2 ± 0.1 P = .0269
Health Today
78.6 ± 1.4
81.0 ± 1.3 P = .0864
85.0 ± 1.2 P < .0001
84.8 ± 1.3 P < .0001
86.0 ± 1.3 P < .0001
86.4 ± 1.6 P = .0024
KOOS
Pain
57.7 ± 1.9
62.7 ± 1.8 P = .0003
68.6 ± 2.0 P < .0001
68.7 ± 2.1 P < .0001
69.0 ± 2.5 P < .0001
68.6 ± 2.8 P = .0003
Symptoms
49.2 ± 1.4
52.2 ± 1.7 P = .0114
55.6 ± 1.6 P < .0001
54.0 ± 1.7 P = .0031
54.4 ± 1.9 P = .0051
56.1 ± 2.2 P = .0020
Activities of Daily Living
66.4 ± 2.0
70.5 ± 2.0 P = .0014
74.6 ± 2.2 P < .0001
75.9 ± 2.2 P < .0001
75.7 ± 2.6 P = .0008
76.4 ± 2.8 P = .0015
Sports & Recreation
37.0 ± 2.2
42.3 ± 2.4 P = .0165
50.3 ± 2.6 P < .0001
50.9 ± 2.6 P < .0001
51.8 ± 3.0 P < .0001
52.2 ± 3.2 P < .0001
Quality of Life
37.2 ± 2.0
41.8 ± 2.0 P = .0007
47.7 ± 2.2 P < .0001
48.2 ± 2.3 P < .0001
51.5 ± 2.7 P < .0001
47.6 ± 3.3 P = .0005
SANE
59.6 ± 2.2
69.7 ± 2.1 P < .0001
75.2 ± 2.1 P < .0001
76.6 ± 2.1 P < .0001
77.2 ± 2.3 P < .0001
75.8 ± 2.6 P < .0001
Tegner
3.7 ± 0.2
3.7 ± 0.2 P = .9425
4.2 ± 0.2 P = .0090
4.2 ± 0.2 P = .1018
4.2 ± 0.2 P = .0254
4.5 ± 0.2 P = .0066
VAS
Overall Pain
69.1 ± 3.5
52.4 ± 3.6 P < .0001
42.8 ± 3.4 P < .0001
41.6 ± 3.7 P < .0001
40.8 ± 4.1 P < .0001
38.4 ± 4.4 P < .0001
Strenuous Work Pain
88.6 ± 3.7
74.0 ± 3.9 P < .0001
64.5 ± 4.0 P < .0001
62.0 ± 4.4 P < .0001
58.1 ± 4.6 P < .0001
56.8 ± 5.2 P < .0001
Sedentary Work Pain
38.8 ± 3.3
32.5 ± 3.4 P = .0235
25.9 ± 3.1 P < .0001
24.0 ± 3.1 P < .0001
20.7 ± 2.8 P < .0001
17.4 ± 3.1 P < .0001
Normal Daily Living Pain
58.9 ± 3.7
49.6 ± 3.7 P = .0019
40.4 ± 3.6 P < .0001
34.5 ± 3.5 P < .0001
36.2 ± 4.1 P < .0001
31.6 ± 4.0 P < .0001
Responder Rate
OMERACT-OARSI Simplified
N/A
N/A
N/A
54 (56.8%)
59 (62.1%)
53 (55.8%)
High Improvement
N/A
N/A
N/A
32 (33.7%)
37 (38.9%)
37 (38.9%)
Improvement
N/A
N/A
N/A
50 (52.6%)
54 (56.8%)
46 (48.4%)
NOTE. Mean ± standard error of the mean reported. P values from paired t test compared with baseline.
EQ-5D-5L, EuroQol 5 Dimension 5 Level; KOOS, Knee Injury and Osteoarthritis Outcome Score; N/A, not available; SANE, Single Assessment Numerical Evaluation; VAS, visual analog scale, OMERACT-OARSI, Outcome Measures in Arthritis Clinical Trials—Osteoarthritis Research Society International.
At month 3 postcrossover, 56.8% of patients were considered responders. At months 6 and 12, 62.1% and 55.8% of patients were responders, respectively (Table 4). At 3, 6, and 12 months, 33.7%, 38.9%, and 38.9% of patients were classified as high improvers, respectively.
Temporal Comparison of Original ASA Cohort with Crossover Cohort Following ASA Injection
The visits for both the original ASA (blinded) cohort and the crossover cohort have been time-aligned to the date of ASA injection (Fig 6). The crossover cohort (green line, n = 95) spans 15 months, whereas the original ASA cohort (black line, n = 68) spans the last 12 months of the plot. This temporal shift was done to show that patients who had inadequate relief 3 months after either saline or HA injections had similar responses to patients randomized to ASA when focusing on the time after ASA injection. When comparing the response between cohorts, we found there were no significant differences between the original blinded ASA cohort and the crossover ASA cohort at 3 months, 6 months, or 12 months (P > .05), indicating that prior treatment with HA or saline did not affect subject response from ASA injection.
Fig 6Comparison of response of original ASA randomization cohort (n = 68) versus crossover ASA cohort (n = 95). Average ± standard error of the mean reported for (A) KOOS Pain, (B) KOOS Activities of Daily Living, and (C) VAS Overall Pain. ASA original period = 12 months; ASA crossover group = 15 months. P values for between group changes using PROC MIXED; P > .05 at each time point postinjection. (ASA, amniotic suspension allograft; mo, month; wk, week.)
Immunology data showed no differences between patients who received HA or saline at baseline, 6 weeks, and 6 months for immunoglobulin E, immunoglobulin G, and CRP (P > .05). In the crossover cohort that originally received saline, there were small but statistically significant decreases in immunoglobulin A from baseline to 6 months (180.1 ± 78.3 to 178.7 ± 72.0, P = .0297) and in immunoglobulin M from baseline to 6 weeks (100.7 ± 56.7 to 98.7 ± 56.0, P = .0128). At baseline (before injection), 41.2% (7 patients) tested positive anti-HLA antibodies; whereas 37.7% (26 patients) and 33.8% (25 patients) tested positive at 6 week and 6 months, respectively; there were no statistical differences between time points. TEAEs were also evaluated throughout the study. At 3 months postcrossover, there were no serious TEAEs, 4 moderate TEAEs, which included muscle aching and knee swelling/popping, and 1 mild TEAE, which included arthralgia. At 6- and 12-months postcrossover, there were no severe, moderate, or mild TEAEs reported.
Discussion
In well-matched cohorts compared with ASA randomized subjects, treatment with ASA following failed treatment with HA or saline resulted in significant improvements in the KOOS and the VAS scores compared with crossover baseline at each time point. There were no significant differences in radiographic measures or anti-HLA serum levels compared with baseline, and no severe adverse events reported during the 12-month crossover period. In addition, in this population that previously failed HA or saline injection, more than 55% of patients were responders at months 3, 6, and 12 as measured by the OMERACT-OARSI simplified responder criteria. This manuscript builds upon the growing data evaluating the efficacy of ASA for the treatment of OA; this study evaluates the response of subjects who previously failed HA and saline injections and compares subjects’ blinded response (originally HA or saline) to the combined crossover cohort. Of note, ASA is not on the market as a commercial treatment, but currently under development as a biologic with the Food and Drug Administration. There is a Phase III double-blinded study currently underway (blinded) and there is no available information on the cost of ASA postapproval.
Due to the protocol design for this trial, we had the unique ability to compare PROs from the original randomization period with HA or saline to the crossover period with ASA in the same subjects for the first 3 months of each study period. There is a risk for selection bias due to there not being a strict definition of unacceptable pain at 3 months; however, previous analysis of the data showed that the patients who self-reported unacceptable pain had statistically less improvement at 3 months for KOOS and VAS compared to the patients who remained in the original study.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Furthermore, there is concern that unblinding in this single-arm study could potentially introduce increased placebo effect and additional bias on both investigator and participant side. However, the use of PROs mitigated the occurrence of investigator bias, whereas the establishment of the crossover baseline values for PROs lessened any potential patient bias from their original randomization. Also, patients in this trial reported improvements in PROs that were comparable to those seen in the blinded treatment arm of the original trial, confirming no increased placebo effect due to unblinding.
As reported in the literature, the MID is 12 points for the KOOS Pain, 8-12 points for the KOOS ADL subscores,
Application of minimal important differences in degenerative knee disease outcomes: A systematic review and case study to inform BMJ Rapid Recommendations.
When comparing the original randomization of the crossover cohort, none of the KOOS subscores exceeded the MID, while in the crossover period, the average scores for KOOS Pain and KOOS ADL did. For VAS, in the original randomization, the average score for Overall Pain did not exceed the MID, whereas for the crossover period, the average score for VAS Overall Pain surpassed the higher MID bound (change ≥19.5 mm). In the 200-patient randomized trial, both the KOOS and VAS MIDs were met for all subscores at 3, 6, and 12 months following a single injection of ASA.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
In comparison, a prospective trial using a single injection of triamcinolone acetonide (regular and extended release) exceeded the MID for KOOS QoL at 4-, 8-, 12-, and 24-weeks postinjection,
whereas a prospective case series investigating a single treatment of bone marrow aspirate concentrate showed improvements above the MID in all KOOS subscales 180 days following treatment.
However, a 2-year randomized clinical trial comparing triamcinolone acetonide to saline with injections performed every 3 months showed no significant differences in knee pain and a significant increase in cartilage volume loss in the triamcinolone acetonide group.
Furthermore, a study examining a single injection of adipose-derived mesenchymal stem cells saw more than 67% of patients hit the MID for all the KOOS subscales by 12 months, with an average of 80% for each subscale.
Another interesting variable due to the crossover arm design is whether there was any synergistic effect of treatment with either HA or saline followed by ASA. Due to the study design, the crossover injection (with ASA) was 3 months after the original treatment injection (with HA or saline); when evaluating subject responses across the 15-month study period (Fig 2), pain and function scores showed trends for decreases in responses between 6 weeks and 3 months (crossover baseline), suggesting effects of the original treatment were diminishing. In the original randomization period, the OMERACT-OARSI simplified criteria found that at 3 months, 39.1% of the HA cohort and 44.1% of the saline cohort were considered responders compared with 54.4% of the ASA cohort; in the crossover arm, after receiving an injection of ASA, the responder rate at 3 months was 61.4% for those subjects who originally received HA and 52.9% for those subjects who originally received saline, indicating that prior treatment with HA or saline did not affect subject response from ASA injection.
Breaking down the KOOS scores into the WOMAC subscores based on the Likert scale (stiffness, pain, and function), WOMAC change from baseline for the combined cohort following an injection of ASA at 3 months were –1.0, –1.9, and –6.1 for stiffness, pain, and function, respectively. In a 3-month study comparing celecoxib, a COX-2 inhibitor that was reported to have less side effects than common nonsteroidal anti-inflammatory drugs, researchers found changes in WOMAC score from baseline between –0.9 and –1.2 for stiffness, –2.0 and –3.1 for pain, and –6.8 to –9.5 for function, all which were statistically significant compared to placebo
; the calculated changes to WOMAC in the current study fall within this range. In the current study at 12 months, the percent improvement over the crossover baseline score was 23%, 30%, and 28% for stiffness, pain, and function, respectively, for the combined cohort following ASA treatment.
Another interesting comparison due to the design of the study is the response of the original ASA cohort (blinded) versus the response of the ASA crossover cohort following previous treatment with HA or saline. There were no significant differences between the original and crossover cohorts at any of the time points evaluated, suggesting that previous failed treatment with HA or saline did not significantly impact outcomes following treatment with ASA.
Limitations
This study had limitations: due to the crossover design, all investigators and patients were unblinded to the treatment (ASA). In addition, the study inclusion/exclusion criteria were specific and may not reflect the entire patient population that would benefit from the product. Part of the inclusion criteria was that patients were allowed to have bilateral knee OA; the impact on this study is unknown, but several sources suggest that self-reported measures may be influenced by the presence of pain in the contralateral limb.
Low BMI among the crossover cohort is another limitation of the study. The inclusion criteria required a patient’s BMI to be less than 40; however, the crossover cohort BMI averaged 28, which may not be an accurate representation of the population that could benefit from ASA following prior treatment.
Conclusions
This study showed that patients who previously did not respond to treatment with HA or saline had statistically significant improvements in pain and function scores following a crossover injection of ASA that was sustained for 12 months, as measured by KOOS and VAS. There were no serious adverse events reported, and the injection was safe.
Acknowledgments
We thank Anil Chandraker, M.D., and his laboratory for their work on the anti-HLA testing. The ASA study group includes: Kavita Ahuja, M.D., C.R.C., Catherine Beckham, P.A.-C., James Borchers, M.D., David Shane Buggay, M.D., Jeffrey Clifton, M.D., Ricardo Colberg, M.D., Brian Cole, M.D., M.B.A., Jeffrey Davis, M.D., Kristen Davis, P.A., Jack Farr, M.D., David Flanigan, M.D., Samuel Goldstein, M.D., Andreas Gomoll, M.D., Kenneth Jaffe, M.D., Laith Jazrawi, M.D., Christopher Kaeding, M.D., Christian Lattermann, M.D., Robert Magnussen, M.D., Bert Mandelbaum, M.D., Rebecca McNair, C.R.N.P., Jose Ortega, M.D., Stephanie Pongracz, P.A., Seth Sherman, M.D., Beth Shubin Stein, M.D., Eric Strauss, M.D., Sabrina Strickland, M.D., Samuel Tabet, M.D., Nikhil Verma, M.D., Kathleen Weber, M.D., M.S., William Whitley, Pharm.D., Robert Wilson, M.D., Adam Yanke, M.D., Ph.D., and Kenneth Zaslav, M.D.
A randomized controlled single-blind study demonstrating superiority of amniotic suspension allograft injection over hyaluronic acid and saline control for modification of knee osteoarthritis symptoms.
Safety and efficacy of an amniotic suspension allograft injection over 12 months in a single-blinded, randomized controlled trial for symptomatic osteoarthritis of the knee.
Application of minimal important differences in degenerative knee disease outcomes: A systematic review and case study to inform BMJ Rapid Recommendations.
The authors report the following potential conflicts of interest or sources of funding: A.H.G. reports other from Organogenesis, during the conduct of the study; other from Moximed, JRF, United States, Smith & Nephew, Vericel, Aastrom Biosciences, Aesculap Biologics, Fidia Pharm, Italy, Flexion Therapeutics, Lifenet Health, Stryker, United States, Vericel, and Zimmer Biomet, United States, outside the submitted work. This study was funded by Organogenesis. B.R.M. reports other from Arthrex, outside the submitted work. J.F. reports personal fees, nonfinancial support, and other from Organogenesis, during the conduct of the study; support from Active Implants; personal fees from Aesculap Biologics; other from the American Journal of Orthopedics; personal fees and nonfinancial support from Arthrex, United States; personal fees from Biopoly and CartiHeal; other from Cartilage; personal fees from Cook Biotech; nonfinancial support from Episurf; personal fees from Exactech; nonfinancial support from Fidia Pharma, Italy and Joint Restoration Foundation; other from MedShape; personal fees and nonfinancial support from Moximed; nonfinancial support from Novartis; other from Ortho Regenerative Tech; personal fees from Regentis; personal fees and nonfinancial support from Samumed; other from Springer and Thieme Medical Publishers; personal fees and nonfinancial support from Vericel; nonfinancial support from Zimmer Biomet Holdings; and personal fees from ZKR Orthopaedics, outside the submitted work. W.T.A. reports other from Organogenesis, during the conduct of the study. S.L.S. reports personal fees from Arthrex, BioVentus, Sparta Biomedical, Reparel, NewClip, Ceterix Orthopaedics, Conmed, Flexion Therapeutics, GLG Consulting, Linvatec, Moximed, Olympus, Japan, RTI Surgical, Smith & Nephew, Vericel, Zimmer, Epic Bio, and Joint Restoration Foundation; other from AAOS, ACL Study Group, United States, American Orthopaedic Society for Sports Medicine, United States, Arthroscopy Association of North America, United States, Epic Bio, International Cartilage Regeneration & Joint Preservation Society, International Society of Arthroscopy, Knee Surgery, and Orthopaedic Sports Medicine, Joint Restoration Foundation, Vericel, American Journal of Orthopaedics and Arthroscopy, outside the submitted work. S.K.T. reports personal fees and nonfinancial support from Organogenesis, during the conduct of the study; personal fees and nonfinancial support from Zimmer Biomet, outside the submitted work. K.A.K. and K.C.M. report they are employee of Organogenesis. ICMJE author disclosure forms are available for this article online, as supplementary material.
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