Conditions: Knee Osteoarthritis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 447
Healthy volunteers: no

Brief summary:
The purpose of this study is to determine the safety and effectiveness of micronized dehydrated human amnion chorion membrane as compared to the 0.9% Sodium Chloride Injection, USP placebo control for the treatment of knee osteoarthritis

Detailed description:
Each subject will receive 1 injection (micronized dHACM or placebo) and be evaluated for efficacy and safety during a 12-month observation period. A second injection (open-label access to micronized dHACM) will be offered to all subjects at the 180-day, 270-day, and 365-day time points, it may only be used once. Blinding for the first injection treatment allocation will be maintained until the end of the study.

Subjects who receive the open-label access treatment will continue to be followed for 180 days after this second injection, regardless of the time at which treatment was received.

Arms:
arm 1: Injection of micronized dHACM arm 2: Injection of 0.9% Sodium Chloride Injection, USP

Interventions: intervention 1: Micronized dHACM intervention 2: Saline
Intervention descriptions: intervention 1: Injection of 40 mg micronized dehydrated human amnion chorion membrane (dHACM) suspended in 1 mL 0.9% Sodium Chloride, USP intervention 2: Injection of 1 mL 0.9% Sodium Chloride, USP

Protocol document:
Prot_004.pdf: 
Study Protocol 
 
STUDY NUMBER(S): AIOA001 
 
PROTOCOL(S) TITLE: A PHASE 2B, PROSPECTIVE, DOUBLE-BLINDED, 
RANDOMIZED CONTROLLED TRIAL OF THE MICRONIZED dHACM INJECTION AS 
COMPARED TO SALINE PLACEBO INJECTION IN THE TREATMENT OF 
OSTEOARTHRITIS OF THE KNEE 
 
NCT: 03485157 
 
 
PROTOCOL DATE: 01/31/2020 
 


16.1.1 Protocol 
Confidential
AIOA001 - Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline Placebo 
in Treatment of Knee OA 
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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Document: AIOA001 
Effective Date: 01/31/2020 
Version Number: V2
 
 
SYNOPSIS 
Name of Investigational Product: Allogeneic Micronized Dehydrated Human 
Amnion/Chorion Membrane (Micronized dHACM), a.k.a. AmnioFix Injectable 
Study Design: A Phase 2B, Prospective, Double-Blinded, Randomized Controlled Trial of the 
Micronized dHACM Injection as Compared to Saline Placebo Injection in the Treatment of 
Osteoarthritis of the Knee 
Indication: Knee osteoarthritis (OA) 
Objective: To determine the safety and effectiveness of micronized dHACM as compared to 
the 0.9% Sodium Chloride Injection, USP placebo control for the treatment of knee 
osteoarthritis 
Sample Size: Approximately 466 subjects will be enrolled in this study. Subjects to be 
randomized 1:1 in treatment arms. 
Study Plan: The estimated enrollment period is 26 months. Each subject will receive 1 
injection and be evaluated for efficacy and safety during a 12-month observation period. A 
second injection (open-label access to micronized dHACM) will be offered to all subjects at 
the 180-day, 270-day, and 365-day time points, see open-label access section below; it may 
only be used once. The study is expected to be completed within 44 months, inclusive of 
enrollment and follow-up for all subjects. 
Procedures and Assessments: 
• 
Treatment (approx. 1 day): Screening, pre-procedure evaluation, injection procedure, 
baseline evaluation.  
• 
Clinical Follow-up (365-545 days): Routine follow-up with physical examination; 
pain, function, and quality of life measurements: Visual Analog Scale (VAS) for Pain, 
VAS for Satisfaction, Western Ontario and McMaster Universities (WOMAC) 
osteoarthritis index, and Knee injury and Osteoarthritis Outcome Score (KOOS) will 
be evaluated at baseline, 30 days, 60 days, 90 days, 180 days, 270 days and 365 days 
after the initial treatment, and at time of open-label access and 30 days, 60 days, 90 
days, and 180 days following the second injection should the subject elect to receive 
the open-label access treatment. Subject pain diaries will be completed at predefined 
intervals. 
• 
Safety Follow-up (365-545 days): Adverse Events (AEs), Serious Adverse Events 
(SAEs), and Unanticipated Adverse Events will be evaluated at 30 days, 60 days, 90 
days, 180 days, 270 days and 365 days after the initial treatment, and at time of open-
label access and 30 days, 60 days, 90 days, and 180 days following the second 
injection should the subject elect to receive the open-label access treatment. 
Primary Efficacy Endpoints (VAS for Pain and Total WOMAC):  
The co-primary efficacy endpoints are: 
• 
The change in VAS score for pain between baseline and Day 90 
• 
The change in WOMAC osteoarthritis index total score (Total WOMAC score) 
between baseline and Day 90 
Primary Safety Endpoint: The primary safety endpoint is the proportion of product-related 
Adverse Events (AEs), Serious Adverse Events (SAEs), and unanticipated adverse events 
throughout the study. 
Secondary Endpoints: 
The secondary efficacy endpoints for this study are: 
• 
Change in VAS score for pain between baseline and 180 days 
• 
Change in Total WOMAC score between baseline and 180 days 


16.1.1 Protocol 
Confidential
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in Treatment of Knee OA 
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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Document: AIOA001 
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Exploratory Endpoint: 
For the cohort of subjects in the main phase of the study, i.e. who did not receive or have not 
yet received an open-label injection, the following exploratory endpoints will be assessed: 
• 
Change in VAS score for pain between baseline and i-day (with i=30, 60, 270, and 
365 days) 
• 
Change in Total WOMAC score between baseline and i-day (with i=30, 60, 270, 
and 365 days) 
• 
Change in pain, stiffness, and physical function WOMAC subscale scores between 
baseline and i-day (with i=30, 60, 90, 180, 270, and 365 days) 
• 
Change in VAS score for satisfaction between baseline and i-day (with i=30, 60, 
90, 180, 270, and 365 days) 
• 
Change in KOOS scores between baseline and i-day (with i=30, 60, 90, 180, 270, 
and 365 days) 
• 
Patient pain diary endpoints will be collected and summarized across applicable 
time points 
For the cohort of subjects in the extension phase, i.e. who received the open-label injection and 
are within the period after the open-label injection, the following endpoints will be collected 
and summarized across applicable time points: VAS score for pain, Total WOMAC, WOMAC 
subscales for pain, stiffness and physical function, VAS score for satisfaction, KOOS, and pain 
diary entries. 
Inclusion Criteria  
All subjects enrolled must meet all the following criteria: 
1. Age ≥ 21 and ≤ 80 years 
2. Subject has a diagnosis of osteoarthritis (OA) defined as Grade 1 to 3 on the Kellgren 
Lawrence grading scale 
3. Subject is willing and able provide informed consent and participate in all procedures 
and follow-up evaluations necessary to complete the study 
4. Subject must have a VAS pain scale greater than 45 


16.1.1 Protocol 
Confidential
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Exclusion Criteria  
Any potential subjects meeting any of the following criteria will be excluded from enrollment 
and subsequent randomization. 
1. Subject has a diagnosis of osteoarthritis (OA) defined as Grade 4 on the Kellgren 
Lawrence grading scale 
2. BMI greater than 40 kg/m2 
3. Subject has active infection at the injection site  
4. Symptomatic OA of the contralateral knee or of either hip that is not responsive to 
acetaminophen (Tylenol®) and requires other therapy. 
5. Subject has rheumatoid arthritis, psoriatic arthritis, or have been diagnosed with any 
other disorders that is the primary source of their knee pain, including but not limited 
to: osteonecrosis, radiculopathy, bursitis, tendinitis, tumor, cancer 
6. Subject has documented history of gout or pseudo-gout 
7. Subject has autoimmune disease or a known history of having Acquired 
Immunodeficiency Syndromes (AIDS) or HIV 
8. Subject has received any of the following to the target knee: 
a.  Intra-articular hyaluronic acid (HA) injection within 12 weeks prior to screening 
b. Steroid or platelet rich plasma (PRP) injection within 12 weeks prior to screening 
c. Has had or is planning to have major surgery or arthroscopy in the target knee 
within 26 weeks of treatment  
d. History of a total knee arthroplasty 
9. Subject has used an investigational drug, device or biologic within 12 weeks prior to 
treatment 
10. Subject has a history of immunosuppressive or chemotherapy in the last 5 years 
11. Subject has had prior radiation at the site 
12. Subject is currently taking anticoagulant therapy (excluding Plavix or Aspirin) 
13. Subject is pregnant or plans to become pregnant within 365 days of treatment  
14. Subject has any significant medical condition that, in the opinion of the Investigator, 
would interfere with protocol evaluation and participation 
15. Subject is a worker’s compensation patient 
16. Subject is a prisoner 
Open-Label Access: 
All study subjects will be eligible to receive one injection of micronized dHACM at either of 
the Day 180, Day 270, and Day 365 time points. The investigators and subjects will remain 
blind to the initial treatment assignment at this time, and until the study is unblinded.  
Subjects who receive the open-label access treatment will continue to be followed for 180 days 
after this second injection, regardless of the time at which treatment was received. 
Statistical Methods: 
The change in VAS score for pain and Total WOMAC score between baseline and Day 90 
(primary endpoints) and between baseline and Day 180 (secondary endpoints) will be 
compared between the treatment arms using an ANOVA with treatment as a fixed effect and 
study site as the block effect.  
For the purposes of this protocol, we will be examining knee osteoarthritis. The hypothesis to 
be tested is to confirm that the use of micronized dHACM offers a statistically significant 
advantage over placebo control. 
Efficacy and safety data will be submitted for evaluation following the completion of the study 
follow-up.  

SAP_005.pdf: 
Statistical Analysis Plan (SAP) 
 
STUDY NUMBER(S): AIOA001 
 
PROTOCOL(S) TITLE: A PHASE 2B, PROSPECTIVE, DOUBLE-BLINDED, 
RANDOMIZED CONTROLLED TRIAL OF THE MICRONIZED dHACM INJECTION AS 
COMPARED TO SALINE PLACEBO INJECTION IN THE TREATMENT OF 
OSTEOARTHRITIS OF THE KNEE 
 
NCT: 03485157 
 
 
SAP DATE: 06/24/2020 
 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
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16.1.9 DOCUMENTATION OF STATISTICAL METHODS 
 
 
TABLE OF CONTENTS 


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TABLE OF TABLES 
TABLE OF FIGURES 


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This Statistical Analysis Plan (SAP) is intended to act as an outline for the statistical 
interpretation of the data resulting from the Phase 2B Investigational New Drug (IND) 
trial, titled: “A Phase 2B, Prospective, Double-Blinded, Randomized Controlled Trial of 
the Micronized dHACM Injection as Compared to Saline Placebo Injection in the 
Treatment of Osteoarthritis of the Knee.”  Specific questions regarding this trial’s design 
and/or implementation may be addressed by the study protocol (AIOA001 Protocol V2). 
This study consists of two phases – Main Phase and Open-Label Extension Phase 
(Extension Phase). The Main Phase of the study includes subjects during the observation 
period where they received the randomized treatment only. The Extension Phase of the 
study includes the subjects who elected to receive an open-label injection of micronized 
dHACM injection at either of the 180-day, 270-day or 365-day visits, during the 180-day 
period following the open-label injection.  
In order to facilitate the planning of a Phase 3 pivotal study based on results of the 
primary and secondary efficacy parameters at 90-day and 180-day, the randomized 
treatment for the Main Phase will be unblinded and a snapshot of the database will be 
taken after all randomized subjects completed the 180-day assessment. Formal database 
preparation process will be performed to ensure the integrity and quality of the database 
and an interim analysis of the primary and secondary efficacy endpoints as well as the 
exploratory efficacy endpoints up to the 180-day time point will be performed. At the end 
of the study, the database will be locked. The database lock process will be performed to 
ensure the integrity and quality of the database; and formal analyses will be performed. 
1 STUDY OBJECTIVES 
This Phase 2B IND Trial will be conducted to determine the safety and effectiveness of 
micronized dehydrated Human Amnion/Chorion Membrane (dHACM) as compared to 
the 0.9% sodium chloride, USP placebo control for the treatment of osteoarthritis of the 
knee. 
2 PRIMARY OBJECTIVES 
The primary objective of this study is to determine the effectiveness of pain reduction and 
improvement of function after being treated with micronized dHACM as compared to the 
0.9% sodium chloride, USP placebo control in patients with osteoarthritis of the knee. 
2.1 
Primary Efficacy Endpoints 
The co-primary efficacy endpoints are as follows: 
• Change in Visual Analog Scale (VAS) score for pain between baseline and 90 
days 


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• Change in Western Ontario and McMaster Universities (WOMAC) Osteoarthritis 
Index total score (Total WOMAC score), between baseline and 90 days 
2.1.1 
VAS for Pain 
The VAS for pain1 is a unidimensional measure of pain intensity, which has been widely 
used in diverse adult populations, including those with rheumatic diseases. The VAS for 
pain is a continuous scale comprised of a horizontal visual analog scale, usually 10 
centimeters (100 mm) in length, and is most commonly anchored by “no pain” (score of 
0) and “pain as bad as it could be” or “worst imaginable pain” (score of 100 [100-mm 
scale]). 
2.1.2 
Western Ontario and McMaster Universities (WOMAC) 
Osteoarthritis Index 
The Western Ontario and McMaster Universities (WOMAC) osteoarthritis index2 is a 
validated, self-administered instrument developed in 1982 to evaluate a patient’s 
disability as it relates to arthritis in the knee.  The WOMAC index evaluates the level of 
disability due to osteoarthritis using 24 questions that comprise three subscales: pain, 
stiffness, and function.  The Total WOMAC score is calculated as the sum of each item 
which is on a 0 to 4 point scale.  The items total to a maximum of 96 points with a higher 
score indicating greater disability. 
Should a patient fail to complete all items within a single questionnaire, missing data will 
be handled utilizing recommendations provided within the WOMAC User Guide2, which 
states: 
“If ≥ two pain, both stiffness, or ≥ four physical function items are omitted, the patient’s 
response is regarded as invalid and the deficient subscale(s) should not be used in the 
analysis.  Where one pain, one stiffness, or 1-3 physical function items are missing, we 
suggest substituting the average value for the subscale in lieu of the missing item 
value(s).” 
2.2 
Primary Safety Endpoint 
The primary safety endpoint is the proportion of product-related Adverse Events (AEs), 
Serious Adverse Events (SAEs), and unanticipated adverse events throughout the study. 
3 SECONDARY OBJECTIVES 
The secondary objectives of this study are to determine the longer-term effectiveness of 
pain reduction and level of disability after being treated with micronized dHACM as 
compared to the 0.9% sodium chloride, USP placebo control in patients with 
osteoarthritis of the knee. 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
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3.1 
Secondary Efficacy Endpoints 
The secondary efficacy endpoints are as follows: 
• Change in VAS score for pain between baseline and 180 days 
• Change in Total WOMAC score between baseline and 180 days 
4 EXPLORATORY OBJECTIVES 
The exploratory objectives of this study are intended to assess further the effectiveness in 
pain reduction and level of disability after being treated with micronized dHACM as 
compared to the 0.9% sodium chloride, USP placebo control in patients with 
osteoarthritis of the knee, throughout the course of the study. 
As outlined in AIOA001 protocol, all study subjects will become eligible to receive an 
open-label injection of micronized dHACM at either of the 180-day, 270-day, or 365-day 
time points.  Due to this change in treatment from the original protocol, all endpoints 
following the 180-day time point become exploratory objectives.  


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5 STUDY SUCCESS CRITERIA 
6 STUDY DESIGN AND PLAN 


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7 STATISTICAL METHODS 
7.1 
Statistical and Analytical Plans 
7.1.1 
General Considerations 
For the purposes of this protocol, osteoarthritis of the knee outcomes will be examined.  
The hypothesis to be tested is to confirm that the use of micronized dHACM offers a 




16.1.9 Documentation of Statistical Methods 
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7.1.3 
Co-Primary Efficacy Analysis 
This study consists of co-primary efficacy endpoints, which requires both endpoints to 
demonstrate success at 2-sided, 
 level of significance. 
7.1.3.1 
VAS Score for Pain Analysis (90 Days) 
ANOVA on the change in VAS score for pain between baseline and 90 days with 
treatment as a fixed effect and study site as the block effect will be used to compare the 
efficacy between Active and Control groups at 2-sided, 
 level of significance. 
In the case of data not being normally distributed, nonparametric tests, such as Wilcoxon 
Rank Sum test, will be utilized to assess the treatment efficacy. 
7.1.3.2 
Total WOMAC Score Analysis (90 Days) 


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ANOVA on the change in Total WOMAC score between baseline and 90 days with 
treatment as a fixed effect and study site as the block effect will be used to compare the 
efficacy between Active and Control groups at 2-sided, 
 level of significance. 
In the case of data not being normally distributed, nonparametric tests, such as Wilcoxon 
Rank Sum test, will be utilized to assess the treatment efficacy. 
7.1.4 
Secondary Efficacy Analysis 
This study consists of two secondary efficacy endpoints that will be evaluated 
individually to demonstrate success at 2-sided, 
 level of significance.  Success in 
either endpoint is intended to be used for future product claims. 
7.1.4.1 
VAS Score for Pain Analysis (180 Days) 
ANOVA on the change in VAS score for pain between baseline and 180 days with 
treatment as a fixed effect and study site as the block effect will be used to compare the 
efficacy between Active and Control groups at 2-sided, 
 level of significance. 
In the case of data not being normally distributed, nonparametric tests, such as Wilcoxon 
Rank Sum test, will be utilized to assess the treatment efficacy. 
7.1.4.2 
Total WOMAC Score Analysis (180 Days) 


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ANOVA on the change in Total WOMAC score between baseline and 180 days with 
treatment as a fixed effect and study site as the block effect will be used to compare the 
efficacy between Active and Control groups at 2-sided, 
 level of significance. 
In the case of data not being normally distributed, nonparametric tests, such as Wilcoxon 
Rank Sum test, will be utilized to assess the treatment efficacy. 
7.1.5 
Exploratory Analyses 


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7.1.6 
Adjustments for Covariates 
Not applicable. 
7.1.7 
Disposition of Subjects 
The number and percentage of subjects enrolled, randomized, treated, early termination 
in the study will be presented.  Reasons for any screen failures and/or early terminations, 
including the relationship to COVID-19 related study disruption, will be summarized.  In 
addition, the number and percentage of subjects who deviated from the study, including 
the relationship to COVID-19 related study disruption, will also be summarized. 
7.1.8 
Description of Demographics and Baseline Characteristics 
Demographic data, baseline characteristics, medical history, and concomitant 
medications will be summarized by means of descriptive statistics (number, mean, SD, 
median, minimum, and maximum) or frequency tables and analyzed using t-tests, chi-
square tests, or Fisher’s exact tests, as applicable. A listing of subjects affected by the 
COVID-19 related study disruption will be presented. 
7.1.9 
Study Site Assessment 
In addition to adjusting for study site in the primary efficacy analysis, in order to evaluate 
the consistency of efficacy across sites, subgroup analysis by study site will also be 
conducted.  Study sites with less than 10 subjects will be analyzed using pooling 
techniques to provide more reliable inferences across sites. 
7.1.10 
Subgroup Analyses 
Exploratory analysis on efficacy parameters will be conducted on gender, age, BMI, and 
smoking status. 
7.1.11 
Planned Interim Analyses 


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7.1.12 
Multiple Comparisons / Multiplicity 
There will be one interim analysis conducted when the study population reaches 50% of 
the total expected and a second interim analysis conducted after all subjects have 
completed the 180-day visit.  Type I error will be adjusted for the interim analyses as 
mentioned in Section 7.1.11.  Furthermore, one final analysis will be conducted upon 
study completion, which should mitigate any concerns of multiplicity. 
7.1.13 
Handling of Dropouts or Missing Data 
Missing data is expected to be minimal; however, there is a chance that some subjects 
will not have full, complete data for the entire analysis.  Missing data for efficacy 
assessments will be imputed using the Last Observation Carried Forward (LOCF) 
approach.  In conjunction, Observed Case data, reported data without imputation, will 
also be analyzed to assess the sensitivity of the analysis to the choice of imputation 
approach. 
7.2 
Safety Evaluation 
The safety endpoint of this study is the proportion of product-related AEs, SAEs, and 
unanticipated adverse events throughout the study in the micronized dHACM versus 
placebo-treated group. All enrolled subjects who receive at least one injection will be 


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used for the analysis of safety data. A listing of AE affected by the COVID-19 related 
study disruption will be presented. 
The incidence of all treatment emergent adverse events and serious adverse events as 
well as major complications (product related) will be compared between the two 
treatment groups. Safety will also be reported as the frequencies of occurrence of each 
adverse event, the rate of adverse events per subjects/year and time to each event. In 
addition, the number of subjects with each serious adverse event type will be reported. 
7.3 
Changes in the Planned Statistical Methodology 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
Placebo in Treatment of Knee Osteoarthritis 
Confidential
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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7.4 
Presentation of Statistical Tables, Figures, and Data Listings 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
Placebo in Treatment of Knee Osteoarthritis 
Confidential
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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Effective Date: 06/24/2020 
Version Number: V3
 
 
 
 
 
 
 
7.5 
Electronic Datasets 
8 APPENDICES 
None. 
9 REFERENCES 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
Placebo in Treatment of Knee Osteoarthritis 
Confidential
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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10 RELATED DOCUMENTS 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
Placebo in Treatment of Knee Osteoarthritis 
Confidential
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
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Effective Date: 06/24/2020 
Version Number: V3
 
 
 
 
 
 
 
11 REVISION HISTORY 


16.1.9 Documentation of Statistical Methods 
AIOA001 – Prospective, Double-Blinded, RCT of Micronized dHACM vs. Saline 
Placebo in Treatment of Knee Osteoarthritis 
Confidential
Allogeneic Micronized Dehydrated Human Amnion/Chorion Membrane 
Page 27 of 27
 
Document: SAP-AIOA001 
Effective Date: 06/24/2020 
Version Number: V3