A Randomized Controlled Clinical Trial Evaluating the Efficacy of a Unique Advanced Bioengineered Skin Substitute With Standard of Care Versus an Active Comparator With Standard of Care in the Treatment of Non-Healing Diabetic Foot Ulcers
Phase: NA
Conditions: Diabetic Foot Ulcer
Sponsor: Encoll Corp

CONFIDENTIAL 
 
 
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A Randomized Controlled Clinical Trial Evaluating 
The Efficacy Of A Unique Advanced Bioengineered 
Skin Substitute with Standard of Care Versus An 
Active Comparator with Standard of Care In The 
Treatment Of Non-Healing Diabetic Foot Ulcers 
 
 
 
 
 
 
 
 
 
 
Protocol Number: ENC-HEL-DFU-02 
Version: 1.0 
Date: June 7, 2024 
 
 
 
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INVESTIGATOR’S SIGNATURE PAGE 
 
 
 
INVESTIGATOR’S SIGNATURE 
 
 
 
I have read the protocol specified above and agree to participate in and comply with the 
procedures, as outlined herein for the conduct of this clinical trial. I also agree to comply with 
the applicable US Food and Drug Administration (FDA) regulations and Investigational Review 
Board (IRB) requirements for testing on human subjects. I agree to ensure that the 
requirements for obtaining informed consent are met. 
 
 
 
 
 
 
Investigator’s Signature 
Date 
Print Name 
Site Number 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Contents 
LIST OF ABBREVIATIONS ............................................................................................. 7 
PROTOCOL SYNOPSIS: ENC-HEL-DFU-02 .................................................................. 8 
1 INTRODUCTION ...................................................................................................... 12 
2 RATIONALE FOR STUDY ....................................................................................... 13 
3 STUDY ENDPOINTS ............................................................................................... 14 
4 STUDY DESIGN....................................................................................................... 15 
5 SUBJECT COMPLETION, SUCCESS/FAILURE, WITHDRAWAL AND SCREEN 
FAILURE .................................................................................................................. 26 
5.1 Subject Completion ................................................................................................ 26 
5.2 Success and Failure .............................................................................................. 26 
5.3 Premature Withdrawal from the Study.................................................................... 26 
5.4 Screen Failures ...................................................................................................... 27 
6 STUDY TREATMENT .............................................................................................. 28 
6.1  Method for Assigning Eligible Subjects to Treatment ....................................... 28 
6.2  Helicoll®  Product Information (provided from IFU) .......................................... 28 
6.3  Grafix® Product Information ............................................................................... 30 
6.4  Epifix® Product Information ................................................................................ 32 
7 STANDARD OF CARE, CONCOMITANT MEDICATIONS, EXCLUDED/ALLOWED 
THERAPIES/MEDICATIONS AND ALLOWED DRESSINGS ................................. 35 
7.1 SOC Procedures .................................................................................................... 35 
7.2 Concomitant Medications ....................................................................................... 35 
7.3 Prohibited Medications and Therapies ................................................................... 36 
7.4 Allowable Medications and Therapies .................................................................... 36 
7.5 Approved Dressings ............................................................................................... 37 
8 DESCRIPTION OF PROTOCOL ASSESSMENTS AND PROCEDURES ............... 38 
8.1 Informed Consent .................................................................................................. 38 
8.2 Assessment of Eligibility ......................................................................................... 38 
8.3 Subject Demographics, Medical History and Ulcer History .................................... 38 
8.4 Peripheral Perfusion Assessment .......................................................................... 40 
8.5 Pain Assessment: NPRS ....................................................................................... 41 
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8.6 Ulcer Assessments ................................................................................................ 42 
8.7 Randomization ....................................................................................................... 44 
8.8 Assessment of Offloading ...................................................................................... 44 
8.9 Semmes Weinstein Monofilament Test for Peripheral Neuropathy ........................ 44 
9 DATA AND STATISTICAL ANALYSES .................................................................. 45 
9.1 Data Analysis ......................................................................................................... 45 
The number and type of Treatment Emergent Adverse Events ..................................... 46 
9.2 Statistical Analysis ................................................................................................. 46 
10 ADVERSE EFFECTS AND EVENTS (DEFINITIONS AND REPORTING) .............. 47 
10.1 SOC Adverse Events ............................................................................................. 47 
11 DIRECT ACCESS TO SOURCE DATA/DOCUMENTATION ................................... 50 
12 QUALITY CONTROL AND QUALITY ASSURANCE .............................................. 51 
12.1 Acceptability of CRF .............................................................................................. 51 
12.2 Modification of Protocol .......................................................................................... 51 
12.3 Reporting Protocol Deviations ................................................................................ 51 
13 ETHICS AND REGULATORY REQUIREMENTS .................................................... 52 
13.1 IRB ......................................................................................................................... 52 
13.2 Investigator’s Responsibilities ................................................................................ 52 
13.3 Subject Informed Consent Requirements............................................................... 52 
14 DATA HANDLING AND RECORD KEEPING .......................................................... 54 
14.1 Recording and Collection of Data........................................................................... 54 
14.2 Clinical Data Management and Monitoring ............................................................ 54 
14.3 Archiving ................................................................................................................ 54 
15 PUBLICATION PLAN .............................................................................................. 56 
16 REFERENCES ......................................................................................................... 57 
17. SIGNATURES ......................................................................................................... 58 
Appendix A: Wagner Grades ......................................................................................... 59 
Appendix B: ABI or Arterial Doppler Ultrasound ABI Technique .................................... 60 
Arterial Doppler Ultrasound Technique .......................................................................... 60 
Appendix C: SPP ........................................................................................................... 61 
Appendix D: TCOM Technique....................................................................................... 62 
Appendix E: Debridement .............................................................................................. 63 
 
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TABLE 1: CONTACT INFORMATION 
 
Role 
Contact Information 
Sponsor 
EnColl Corp. 
4576 Enterprise St.  
Fremont, CA 94538 
Website: www.helicoll.com 
 
Sponsor Clinical Contact 
Dr. Subra Guna, PhD 
Phone: 510-396-8581 
Email:  guna@encoll.com 
 
Study Chair 
Charles M Zelen, DPM FACFAS 
Phone: 540-520-5863 
Email: cmzelen@periedu.com 
Study Statistician 
Marissa Carter, PhD 
Phone: 307-272-9906 
Email: mcarter@strategic-solutions-inc.com 
Clinical Research 
Organization 
Professional Education and Research Institute 
Jillian Boertlein 
Phone: 513-882-8483  
Email: Jboertlein@periedu.com 
 
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TABLE 2: VERSION HISTORY 
 
Version 
Number 
Release Date 
Change(s) 
Reason for Change 
1.0 
June 7, 2024 
NA 
NA 
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LIST OF ABBREVIATIONS 
 
TERM 
DEFINITION 
ABI/ABPI 
Ankle/Brachial Index/Ankle Brachial Pressure Index 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DFU 
Diabetic foot ulcer 
EOS 
End of study 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HCV 
Healing Confirmation Visit 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency virus 
IRB 
Institutional Review Board 
ITT 
Intent-to-treat 
PAR 
Percentage area reduction 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SOC 
Standard of care 
SOP 
Standard operating procedure 
SPP 
Skin perfusion pressure 
SV 
Screening visit 
TCOM 
Transcutaneous oxygen measurement 
TV 
Treatment Visit 
US 
United States of America 
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PROTOCOL SYNOPSIS: ENC-HEL-DFU-02 
 
Objective 
The purpose of this clinical evaluation is to collect patient outcome data on a 
commercially available 510K FDA cleared advanced skin substitute. The 
commercially available product is Helicoll® Advanced Skin Substitute. 
In this trial two groups of subjects with Wagner 1 diabetic foot ulcers (DFUs), 
will receive standard of care (SOC) treatment for their condition.  Half of the 
patients will have their SOC treatment with Epifix® or Grafix® and the other 
half will receive a 510K FDA cleared Helicoll® Advanced Skin Substitute as 
the primary treatment. The primary endpoint is the percentage wound area 
reduction of the target ulcer. Secondary endpoints include the proportion of 
subjects that obtain complete closure over the 5-week treatment period, the 
time to achieve complete wound closure of the target ulcer by the end of 5 
weeks, and mean number of IP applications. 
Intervention 
Groups 
Group 1: SOC primary dressing with Helicoll®  
Group 2: SOC primary dressing with Epifix® or Grafix®  
Study 
Design 
Multi-center, open label, randomized controlled trial 
Sample Size 
For this RCT, N=24 subjects completed 
Centers 
Up to 5 centers within the United States, Open/competitive enrollment 
Endpoints 
Primary endpoint: 
- 
Percentage wound area reduction from TV1 to TV5 measured 
manually with digital photography 
 Secondary endpoints: 
- 
The time to achieve complete wound closure of the target ulcer by 
the end of 5 weeks 
- 
The proportion of subjects that obtain complete closure over the 5-
week treatment period  
- 
Mean number of repeated applications of the Advanced Skin 
Substitute used to obtain wound closure 
Exploratory Endpoint: 
- 
The appearance, structural stability, and fragility of the new 
skin formed documented at each visit.  Any recurrence of the 
wound also will be monitored. 
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Safety Analysis: 
- 
The number and type of Treatment Emergent Adverse and Serious 
Adverse Events 
Safety 
There is no formal safety objective for this study as Helicoll®,  Epifix® and 
Grafix® are commercially available, 510K FDA cleared products for 
application on diabetic foot wounds.  However, there is the possibility of 
disease transmission due to the risk of viral infection upon application of an 
allograft-tissue-derived skin substitute. Therefore, in accordance with FDA 
regulations, adverse reactions to the product will be recorded and provided to 
the study Sponsor for required FDA reporting and will be concurrently 
reported to all Investigators and IRBs. 
Adverse events associated with the application of standard of care dressing 
will be collected during the course of the study. 
Collection and reporting of any adverse reaction data, in accordance with FDA 
regulations and adverse events associated with product application will 
ensure study subject safety. 
Surveillance 
Schedule 
Subjects will undergo a screening visit to determine eligibility. Eligible 
subjects will then undergo up to a 5-week treatment phase involving weekly 
evaluations over 5 weeks and after 5 weeks, will exit the study.  Additional 
weekly visits will occur during the 2nd and 3rd week.   
Study 
Duration 
It is estimated that about 3 months will be required to complete the study with 
data collection and reporting. 
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Inclusion 
Criteria 
Potential subjects are required to meet all the following criteria for 
randomization into the study. 
1. Subjects must be at least 18 years of age or older. 
2. Subjects must have a diagnosis of type 1 or 2 Diabetes mellitus. 
3. At randomization subjects must have a target diabetic foot ulcer with a 
minimum surface area of 1.0 cm2 and a maximum surface area of 10.0 
cm2 measured post debridement using a ruler to measure wound area. 
4. The target ulcer must have been present for a minimum of 4 weeks and 
a maximum of 52 weeks of standard of care prior to the initial screening 
visit. 
5. The target ulcer must be located on the foot with at least 50% of the 
ulcer below the malleolus. 
6. The target ulcer must be full thickness on the foot or ankle that does not 
probe to bone. 
7. Adequate circulation to the affected foot as documented by any of the 
following methods performed within 3 months of the first screening 
visit: 
a. TCOM ≥30 mmHg 
b. ABI between 0.7 and 1.3 
c. PVR: Biphasic 
d. TBI ˃0.6 
e. As an alternative, arterial Doppler ultrasound can be performed 
evaluating for biphasic dorsalis pedis and posterior tibial 
vessels at the level of the ankle of the target extremity. 
8. If the subject has two or more ulcers, they must be separated by at least 
2 cm. The largest ulcer satisfying the inclusion and exclusion criteria will 
be designated as the target ulcer. 
9. The subject must consent to using the prescribed off-loading method for 
the duration of the study. 
10. The subject must agree to attend the twice-weekly/weekly study visits 
required by the protocol. 
11. The subject must be willing and able to participate in the informed 
consent process. 
12. Patients must have read and signed the IRB approved ICF before 
screening procedures are undertaken. 
 
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Exclusion 
Criteria 
Exclusion Criteria: 
Potential subjects meeting any of the following criteria will be excluded from 
randomization. 
1. A subject known to have a life expectancy of <6 months. 
2. If the target ulcer is infected or if there is cellulitis in the surrounding skin. 
3. Presence of osteomyelitis or exposed bone, probes to bone or joint 
capsule on investigator’s exam or radiographic evidence. 
4. A subject that has an infection in the target ulcer that requires systemic 
antibiotic therapy. 
5. A 
subject 
receiving 
immunosuppressants 
(including 
systemic 
corticosteroids at doses greater than 10 mg of Prednisone per day or 
equivalent) or cytotoxic chemotherapy. 
6. Topical application of steroids to the ulcer surface within one month of 
initial screening. 
7. A subject with a previous partial amputation on the affected foot is 
excluded if the resulting deformity impedes proper offloading of the 
target ulcer. 
8. A subject with a glycated hemoglobin (HbA1c) greater than or equal to 
13% taken at or within 3 months of the initial screening visit. 
9. A subject with a serum creatinine ≥ 3.0mg/dL within 6 months 
of the initial screening visit. 
10.  A subject with an acute Charcot foot, or an inactive Charcot foot, that 
impedes proper offloading of the target ulcer. 
11. Women who are pregnant or considering becoming pregnant within the 
next 6 months. 
12. A subject with end stage renal disease requiring dialysis. 
13. A subject who participated in a clinical trial involving treatment with an 
investigational product within the previous 30 days. 
14. A subject who, in the opinion of the Investigator, has a medical or 
psychological condition that may interfere with study assessments. 
15. A subject treated with hyperbaric oxygen therapy or a Cellular and/or 
Tissue Product (CTP) in the 30 days prior to the initial screening visit. 
16. A subject who has a sensitivity to bovine (cattle) or ovine (sheep) 
material. 
17. A subject that is allergic to aminoglycoside antibiotics (gentamycin, 
tobramycin, etc.) 
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1 
INTRODUCTION 
1.1 Costs of Non-healing Diabetic Foot Ulcers 
The economic burden of DFUs costs the United States over $50 billion each year.1 The cost 
to treat one patient with DFUs ranges from $11,700 to $16,883.2 Although approximately 
70% of DFUs are shown to heal with good SOC, at least 30% become chronic wounds.3 
These non-healing wounds are at greater risk for infection and lower extremity amputation.4 
Consequently, improved treatment options are important for patients with chronic DFUs to 
improve patient outcomes, lower treatment costs and reduce the risk of complications. 
1.2 Helicoll®  Advanced Skin Substitute  
Helicoll® Semi-Occlusive, Self-Adhering and Sterilized Type-I Collagen Sheet for Wound 
Treatments and Chronic Ulcers. 
DESCRIPTION OF THE DEVICE 
Helicoll® is a translucent, off-white, semi-occlusive, self-adhering and pre-sterilized 
Type-I Collagen Sheet for use as a bioactive membrane. Helicoll® is flexible with 
moderate tackiness. Helicoll® is a reconstituted collagen sheet free of contaminants 
like lipids, elastin, and other immunogenic proteins (US Patented). 
Helicoll® is 510K cleared (K040314) for use in diabetic foot ulcers and a range of other 
acute and chronic wounds.
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2 
RATIONALE FOR STUDY 
Helicoll® is an advanced skin substitute device that is 510K cleared for application on 
diabetic foot wounds and has been shown in case studies and clinical practice to assist in 
wound healing. Therefore, based on this early promising data, a 24 subject RCT is 
necessary to further validate these case studies and identify the likelihood of wound healing 
with weekly or as needed application. For consistency, one type of wound, Wagner 1, will be 
studied in this trial and DFUs have been chosen as they are some of the most common 
wounds seen in wound clinics. 
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3 
STUDY ENDPOINTS 
The purpose of this clinical evaluation is to collect patient outcome data on 2 commercially 
available standard of care dressings in partial/full-thickness diabetic foot ulcer subjects. 
Patient outcomes will be compared at 5 weeks. 
 
The primary endpoint is the percentage wound area reduction from TV1 to TV5 measured 
manually with digital photography. 
 
Secondary endpoints include a comparison between groups of the following clinical measures:   
1. The time to achieve complete wound closure of the target ulcer by the end of 5 weeks. 
2. The proportion of subjects that obtain complete closure over the 5-week treatment period.  
3. Mean number of repeated applications of the Advanced Skin Substitute used to obtain 
wound closure. 
Exploratory Endpoint: 
1. The appearance, structural stability, and fragility of the new skin formed 
documented at each visit.  Any recurrence of the wound also will be monitored. 
 
Safety Analysis: 
1.  The number and type of Treatment Emergent Adverse and Serious Adverse Events.
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4 
STUDY DESIGN 
This study is a prospective, multi-center, RCT designed to collect patient outcome data on 2 
commercially available SOC dressings for the treatment of DFUs.   
There are two standard of care arms in the study: 
 
Arm 1: The SOC therapy in this study is offloading of the DFU (CAM boots or total contact 
casting [TCC] if the subject’s foot is too large for a CAM boot), appropriate sharp or surgical 
debridement, infection management (systemic antibiotics only in conjunction with 
debridement) and wound care covering with Helicoll® applied weekly or as needed followed 
by a padded 3-layer dressing comprised of first layer - non-adherent and porous, second 
layer – absorbent 4x4 gauze pads & third layer - soft roll and compressive wrap (Dynaflex™ 
or equivalent). 
 
Arm 2: The SOC therapy in this study is offloading of the DFU (CAM boots or total contact 
casting [TCC] if the subject’s foot is too large for a CAM boot), appropriate sharp or surgical 
debridement, infection management (systemic antibiotics only in conjunction with 
debridement) and wound care covering with Epifix® or Grafix® followed by a padded 3-layer 
dressing comprised of first layer - non-adherent and porous, second layer – absorbent 4x4 
gauze pads & third layer - soft roll and compressive wrap (Dynaflex™ or equivalent). 
The study involves two phases: Screening and Treatment. 
4.1 Phase 1: Screening 
The Screening Phase is designed to determine whether subjects are eligible to proceed to 
the Treatment Phase of the study and consists of a series of screening assessments 
designed to determine eligibility. 
At the Screening Phase Visit (SV1), written informed consent (ICF) from the subject will be 
obtained by the Investigator or suitably qualified designee before any protected health 
information is obtained and prior to the performance of any protocol-specific procedures. 
After obtaining ICF, the Investigator will select the study target ulcer. Each subject will have 
only one DFU selected as the target ulcer. If the subject has more than one DFU at the SV1 
visit, the Investigator will select the largest DFU that meets the eligibility criteria of the 
protocol as the target ulcer. If the subject meets all eligibility criteria at the Screening Visit, 
then the Screening Visit and first Treatment visit may occur on the same day.     
4.2 Phase 2: Treatment 
The Treatment Phase (up to 5 weeks) begins with a series of assessments designed to 
confirm the subject’s continued eligibility. Investigators will debride the target ulcer, in 
accordance with SOC if required. Subjects whose ulcers continue to meet eligibility criteria 
will then be randomized to one of two groups: (1) SOC with Helicoll® primary dressing 
applied weekly or as needed per investigator discretion or (2) SOC with Epifix® or Grafix® 
applied weekly or as needed per investigator discretion.  
 
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4.3 Subject Treatment Assessments 
4.3.1 DFU Assessments 
During the Treatment Phase, subjects will be evaluated on a weekly basis, with the 
exception of week 2 and 3 where the patient will be seen twice weekly. Weekly/Twice-
Weekly patient outcome evaluations include the Investigator’s assessment of target ulcer 
healing and measurements of ulcer size using a ruler to measure wound area and provide 
photos of wounds, including photos of healed wounds. 
Note: All procedures required during Screening are included in TV1 prior to the first 
treatment visit if the subject fulfills eligibility criteria. 
Other evaluations during the Treatment Phase are detailed in the procedures section. 
Subjects will be seen weekly (± 2 days) until the ulcer is healed or they meet protocol criteria 
to exit the study with the exception of the 2nd and 3rd week of treatment where the subject will 
be seen twice.  For both groups, assigned treatment will be applied only once during the 2nd 
and 3rd week unless the investigator feels an additional application is necessary. If the 
additional application is not necessary, the additional visit will involve a dressing change and 
wound evaluation.  
4.3.2 Wound Healing Assessment 
Initial and confirmation evaluations of wound healing will be subject to oversight by the study 
chair who will review all photographs of healed wounds and confirm wound healing.  
4.4 Study Subjects 
This prospective randomized trial will randomize 24 subjects in up to 5 centers located in the 
USA. 
4.4.1 Inclusion Criteria 
Potential patients are required to meet all the following criteria for randomization into the 
study: 
1. Subjects must be at least 18 years of age or older. 
2. Subjects must have a diagnosis of type 1 or 2 Diabetes mellitus. 
3. At randomization, subjects must have a target diabetic foot ulcer with a minimum 
surface area of 1.0 cm2 and a maximum surface area of 10.0 cm2 measured post 
debridement using a ruler to measure wound area. 
4. The target ulcer must have been present for a minimum of 4 weeks and a maximum of 52 
weeks of standard of care prior to the initial screening visit. 
5. The target ulcer must be located on the foot with at least 50% of the ulcer below the 
malleolus. 
6. The target ulcer must be full thickness on the foot or ankle that does not probe to bone. 
7. Adequate circulation to the affected foot as documented by any of the following 
methods performed within 3 months of the first screening visit: 
a. TCOM ≥30 mmHg 
b. ABI between 0.7 and 1.3 
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c. PVR: Biphasic 
d. TBI ˃0.6 
e. As an alternative, arterial Doppler ultrasound can be performed evaluating 
for biphasic dorsalis pedis and posterior tibial vessels at the level of the 
ankle of the target extremity. 
8. If the subject has two or more ulcers, they must be separated by at least 2 cm. The 
largest ulcer satisfying the inclusion and exclusion criteria will be designated as the 
target ulcer. 
9. The subject must consent to using the prescribed off-loading method for the duration of 
the study. 
10. The subject must agree to attend the twice-weekly/weekly study visits required by the 
protocol. 
11. The subject must be willing and able to participate in the informed consent process. 
12. Patients must have read and signed the IRB approved ICF before screening procedures are 
undertaken. 
4.4.2 Exclusion Criteria 
Potential subjects meeting any of the following criteria will be excluded from randomization: 
1. A subject known to have a life expectancy of < 6 months. 
2. If the target ulcer is infected or if there is cellulitis in the surrounding skin. 
3. Presence of osteomyelitis or exposed bone, probes to bone or joint capsule on 
investigator’s exam or radiographic evidence. 
4. A subject has an infection in the target ulcer or in a remote location that requires 
systemic antibiotic therapy. 
5. A subject receiving immunosuppressants (including systemic corticosteroids at 
doses greater than 10 mg of Prednisone per day or equivalent) or cytotoxic 
chemotherapy. 
6. Topical application of steroids to the ulcer surface within one month of initial 
screening. 
7. A subject with a previous partial amputation on the affected foot is excluded if the 
resulting deformity impedes proper offloading of the target ulcer. 
8. A subject with a glycated hemoglobin (HbA1c) greater than or equal to 13% taken 
at or within 3 months of the initial screening visit. 
9. A subject with a serum creatinine ≥ 3.0mg/dL within 6 months of the initial 
screening visit. 
10. A subject with an acute Charcot foot, or an inactive Charcot foot, that impedes 
proper offloading of the target ulcer. 
11. Women who are pregnant or considering becoming pregnant within the next 6 
months. 
12. A subject with end stage renal disease requiring dialysis. 
13. A subject who participated in a clinical trial involving treatment with an investigational 
product within the previous 30 days. 
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14. A subject who, in the opinion of the Investigator, has a medical or psychological 
condition that may interfere with study assessments. 
15.  A subject treated with hyperbaric oxygen therapy or a Cellular and/or Tissue 
Product (CTP) in the 30 days prior to the initial screening visit. 
16. A subject who has a sensitivity to bovine (cattle) or ovine (sheep) material. 
17. A subject that is allergic to aminoglycoside antibiotics (gentamycin, 
tobramycin, etc.) 
 
4.5 Study Schedule 
The study is divided into two phases: Screening and Treatment Phases. The schedules for 
the protocol-specified assessments and procedures in each phase are detailed in the 
following sections. Note that there is an end of study visit (EOS1) scheduled on Week 5 or 
earlier if the wound heals.   
4.5.1 Visit Windows 
Subject visit dates must be scheduled within the visit windows detailed in the Schedule of Study 
Visits table. When determining visit dates, the reference should always be seven days (+/- 2 
days), except for TV1 which can be +2 days from SV1 or occur on the same day as SV1.  Every 
attempt should be made to maintain subjects on their original treatment schedule. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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TABLE 1: SCHEDULE OF VISITS. LIGHT GREY (SCREENING); LIGHT YELLOW (TREATMENT); LIGHT BLUE (END OF TREATMENT VISIT)  
 
Visit 
SV1 
Wk 1 TV1 
Wk 2 TV2 
Wk 3 TV3 
Wk 4 
TV4 
Wk 5 [C] 
EOS 1 
Pre** 
Rand 
Post 
Rand 
Weeks from 
Treatment Date 
0 
 
0 
1 
 
1.5 
2 
 
2.5 
3 
4 
Window Period 
 
+2 days 
±2 
days 
+2 days 
±2 
days 
+2 days 
±2 
days 
±2 
days 
Assessment of 
eligibility 
X 
X 
Informed consent 
X 
Ulcer history 
X 
Demographics 
X 
Medical History or 
changes 
X 
X 
X 
 
X 
 
X 
X 
Prohibited Therapies 
assessment 
X 
X 
X 
X 
X 
X 
Concomitant 
Medication assessment 
X 
X 
X 
X 
X 
X 
ABI/SPP/TCOM/ 
TBI or Arterial Doppler 
study 
X 
 
X 
 
X 
 
X 
Randomization 
X 
Ulcer assessments 
X 
X 
X 
X 
X 
X 
X 
X 
Infection assessment of 
target ulcer 
X 
X 
X 
 
X 
 
X 
 
X 
X 
X [B] 
Physical exam or 
changes 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
Assess for Adverse Effects 
and Adverse 
Events 
 
X 
X 
X 
X 
X 
X 
X 
Urine or blood pregnancy 
test control (females of 
childbearing 
potential) 
 
 
X 
 
 
 
 
 
 
 
 
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Visit 
SV1 
Wk 1 TV1 
Wk 2 TV2 
Wk 3 TV3 
Wk 4 
TV4 
Wk 5 [C] 
EOS 1 
Pre** 
Rand 
Post 
Rand 
Weeks from 
Treatment Date 
 
0 
 
0 
 
1 
 
1.5 
 
2 
 
2.5 
 
3 
 
4 
Assurance of effective 
birth control (females of 
childbearing 
potential) 
X 
Pain assessment 
X 
X 
X 
X 
X 
X 
X 
X 
Monofilament 
Test (10-point) 
X 
X 
X 
X 
X 
X 
X 
X 
Serum Creatinine 
X 
Blood Glucose 
X 
X 
X 
X 
X 
X 
X 
X 
HbA1c Lab 
X 
X 
Take X-ray to rule 
out osteomyelitis or bone 
infection 
X 
Target ulcer photographs 
X 
X 
X 
X 
X 
X 
X 
X 
Target ulcer measurements 
X 
X 
X 
X 
X 
X 
X 
X 
Target ulcer cleaning 
X 
X 
X 
 
X 
 
X 
X [B] 
Target ulcer debridement 
X[A] 
X[A] 
X[A] 
 
X[A] 
 
X[A] 
X[A] [B] 
Target ulcer closure 
assessment 
X 
X 
 
X 
 
X 
 
X 
X 
X 
Assessment of 
offloading 
X 
X 
X 
X 
X 
X 
X 
Apply 
Helicoll® or Epifix® or 
Grafix®  
X 
X 
 
X 
 
X 
 
Initiate offloading of the 
target ulcer 
X 
 
 
 
 
 
 
 
Apply outer 
dressings 
X 
X 
X 
X 
X 
X 
X 
X[B] 
[A]: If required per guidelines; [B] Only if subject is not healed; [C]: Will be earlier if wound heals before this date or subject is withdrawn from the 
study  
 
**Pre-randomization assessments are only performed if treatment is not done on the same day as screening. Otherwise, Screening and 
Randomization (first treatment) can occur on the same day, screening procedures are performed, then followed by randomization and completion 
of post randomization schema of events.
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4.5.2 Screening Phase 
The subject will sign and date the ICF and HIPAA authorization (according to site practices) prior to 
collection of any protected health information, as well as any study-related procedures.  A subject 
ID will be assigned to each subject in successive order of entering the study after signing the ICF, 
beginning with 001 at each site. 
SV1 can occur up to 2 days prior to randomization.  However, subjects who meet all eligibility 
criteria can enter the Treatment Phase immediately once all of the eligibility criteria are reviewed 
and the subject meets all inclusion and no exclusion criteria. 
4.5.3 SV1: Procedures to be performed include: 
▪ 
Informed consent will be obtained prior to the collection of any protected health information 
as well as any study related procedures and the subject will sign a written ICF 
▪ 
Assess current wound therapies for prohibited therapies within 30 days of randomization 
▪ 
Demographics 
▪ 
Medical history 
▪ 
Physical examination 
▪ 
Assess concomitant medications 
▪ 
Vital signs 
▪ 
Pain assessment (see section 8.5) 
▪ 
Labs: HbA1c (if no documented results within 90 Days of the first screening visit are 
available), assess blood glucose, and serum creatinine (if no documented results within 6 
months of the first screening visit are available) 
▪ 
Pregnancy test (blood or urine) if female subject is of childbearing potential. If female is 
not of childbearing potential the reason for inclusion must be included in the study record 
▪ 
Assurance of effective birth control if female subject is of childbearing potential 
▪ 
Ulcer history 
▪ 
Target Ulcer assessment 
▪ 
Assess signs and symptoms for clinical infection of the target ulcer (see section 8.6.3) 
▪ 
Target ulcer foot neuropathy Semmes Weinstein 10-point test; (see section 8.9) 
▪ 
Clean the target ulcer 
▪ 
Debridement of the target ulcer, if applicable (see Appendix E) 
▪ 
Digital imaging of the target ulcer 
▪ 
Record target ulcer measurements 
▪ 
Obtain X-ray to rule out osteomyelitis or bone infection of the affected foot 
▪ 
ABI, SPP, TCOM, TBI, PVR measurement or Arterial Doppler Study will be obtained (see 
Appendices B-D) 
▪ 
Initiate offloading of the target ulcer 
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▪ 
Assessment of subject’s eligibility to continue in the study 
▪ 
Apply appropriate SOC to target ulcer and outer dressing if patient does not enter into the treatment 
phase immediately following SV1 
4.5.4 Treatment Phase 
When determining the visit dates, the reference should always be seven days (±2 days). 
Every attempt should be made to maintain subjects on their original treatment schedule. 
4.5.4.1 TV1: Pre-Randomization 
The Treatment Phase begins with a series of assessments designed to confirm the subject’s 
continued eligibility (Pre-randomization, Table 1).  The following assessments and activities are 
performed at this visit (unless randomization is performed at the same visit as screening): 
▪ 
Medical history changes from previous visit 
▪ 
Physical examination changes from previous visit 
▪ 
Assess concomitant medications, changes from previous visit and prohibited therapies 
▪ 
Vital Signs 
▪ 
Pain assessment (see section 8.5) 
▪ 
Assess blood glucose 
▪ 
Target ulcer Assessments 
▪ 
Assess signs and symptoms for clinical infection of the target ulcer (see section 8.6.3) 
▪ 
Target ulcer foot neuropathy Semmes Weinstein 10-point test; (see section 8.9) 
▪ 
Clean the target ulcer 
▪ 
Debridement of the target ulcer if applicable (see Appendix E) 
▪ 
Digital imaging of the target ulcer 
▪ 
Record target ulcer measurements 
▪ 
Assessment of offloading 
▪ 
Target ulcer closure assessment will be determined by a site Investigator 
▪ If the subject is seen on a subsequent visit to enter the treatment phase and the 
target ulcer is 100% re-epithelialized, the subject will be considered a screen 
failure. 
▪ 
Assessment of subject’s eligibility to continue in the study 
▪ 
If the subject is eligible, they will enter the treatment phase  
▪ 
If the subject is not eligible, discharge the subject from the study as a screen failure 
 
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4.5.4.2 TV1: Randomization 
The following assessments and activities are performed at this visit: 
▪ 
Randomize the subject 
▪ 
Apply appropriate treatment to target ulcer: 
▪ 
Apply SOC therapy with Helicoll® - Group 1 
▪ 
Apply SOC therapy with Epifix® or Grafix® - Group 2 
▪ 
Apply outer layer dressings, including first layer - non-adherent and porous, second layer – 
absorbent 4x4 gauze pads & third layer - soft roll and compressive wrap (Dynaflex™ or equivalent).  
▪ 
Check for any changes in the subject’s health and assess them for any adverse effects 
and adverse events 
▪ 
Ensure offloading of the wound is satisfactory 
▪ 
Schedule the next study visit for one week later (±2 days) , except TV2 and TV3 where the patient 
will be seen twice weekly.  
4.5.4.3 Treatment Phase Visits: Weeks 2-4                                                                     
The following assessments and activities are performed at these visits: 
▪ 
Medical history changes from previous visit 
▪ 
Physical examination changes from previous visit 
▪ 
Assess concomitant medications, changes from previous visit and prohibited therapies 
▪ 
ABI, SPP, TCOM, TBI, PVR measurement or Arterial Doppler Study will be obtained (see 
Appendices B-D) 
▪ 
Vital Signs 
▪ 
Pain assessment (see section 8.5) 
▪ 
Assess blood glucose 
▪ 
Assessment of offloading 
▪ 
Target ulcer foot neuropathy Semmes Weinstein 10-point test; (see section 8.9) 
▪ 
Check for any changes in the subject’s health and assess them for any adverse effects 
and adverse events 
▪ 
Target ulcer closure assessment will be determined by a site Investigator 
▪ 
If the target ulcer is 100% re-epithelialized, the subject will exit study at the 
conclusion of the visit (the visit will be considered EOS1). Please perform all 
applicable assessments and study procedures associated with EOS1. 
▪ 
Target ulcer Assessments 
▪ 
Check for signs of clinical infection. If a clinical diagnosis of infection has been made, then the 
subject can be treated with oral antibiotics, but topical antibiotics or antimicrobial dressings 
CANNOT be used on the target ulcer (see section 8.6.3) 
▪ 
Clean the target ulcer 
 
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▪ 
Debridement of the target ulcer if applicable (see Appendix E) 
▪ 
Digital imaging of the target ulcer 
▪ 
Record target ulcer measurements 
▪ 
Apply appropriate treatment to the index ulcer: 
▪ 
Apply SOC therapy with Helicoll® - Group 1 
▪ 
Apply SOC therapy with Epifix® or Grafix® - Group 2 
▪ 
For the TV2 and TV3 additional visit where application of a new Helicoll® does not 
occur, the primary dressing will be lifted from the wound site along with the Helicoll® 
Advanced Skin Substitute or Epifix® or Grafix®.  If the Helicoll® Advanced Skin Substitute 
is adherent to the wound, moisten gently with saline before attempting to remove. 
Cleaning of the target ulcer as noted above is suggested, however debridement should be 
limited, and it is suggested that only a very gentle removal of any slough be performed at 
the investigator discretion. Digital Photography is then taken and the Helicoll® Advanced 
Skin Substitute or Epifix® or Grafix® and primary dressing should be placed back on the 
wound. If the primary dressing is soiled or damaged a new primary dressing can be 
applied. Additional assessments will be performed as referenced in the Schedule of Visits 
(Section 4.5, Table 1). 
▪ 
Ensure offloading of the Target ulcer is satisfactory 
▪ 
Apply outer layer dressings, including first layer - non-adherent and porous, second layer – 
absorbent 4x4 gauze pads & third layer - soft roll and compressive wrap (Dynaflex™ or equivalent).  
▪ 
The next visit will be scheduled for one week (+/- 2 days) 
4.5.5 End of Study (EOS1) Visit 
The following assessments and activities are performed at this visit: 
▪ 
Medical history changes from previous visit 
▪ 
Physical examination changes from previous visit 
▪ 
Assess concomitant medications, changes from previous visit and prohibited therapies 
▪ 
Vital Signs 
▪ 
Pain assessment (see section 8.5) 
▪ 
Assess Blood glucose 
▪ 
HbA1c 
▪ 
Assessment of offloading 
▪ 
Target ulcer foot neuropathy Semmes Weinstein 10-point test; (see section 8.9) 
▪ 
Check for any changes in the subject’s health and assess them for any adverse effects 
and adverse events 
▪ 
Target ulcer closure assessment will be determined by a site Investigator 
▪ 
Target Ulcer Assessments 
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▪ 
Check for signs of clinical infection. If a clinical diagnosis of infection has been made, then 
the subject can be treated with oral antibiotics, but topical antibiotics or antimicrobial 
dressings CANNOT be used on the study ulcer 
▪ 
Clean the target ulcer 
▪ 
Debridement of the target ulcer if applicable (see Appendix E) 
▪ 
Digital imaging of the target ulcer 
▪ 
Record target ulcer measurements 
▪ 
Ensure outer dressings are applied and if applicable, make an appointment for the 
subject with their desired clinician for follow-up care 
4.5.6 Unscheduled Visits 
Unscheduled visits may be required in addition to the visits detailed above. Additional visits 
are at the discretion of the Investigator. An example of an unscheduled visit is when a change 
in dressings is required between scheduled visits. The details of these unscheduled visits with 
subjects will be recorded in the medical records/source documents and on the Unscheduled 
Visit CRF. 
4.5.7 
Missed Visits 
If a subject misses a visit, the site is to make every effort to have the subject return as soon as 
possible to make up the visit. Once the subject is seen, he/she is to return to his/her original 
weekly visit schedule. For example, if a subject was seen regularly on Mondays but missed a 
scheduled Monday visit and came in on Wednesday, he/she should return the next Monday to 
maintain his/her weekly Monday visit schedule. 
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5 
SUBJECT COMPLETION, SUCCESS/FAILURE, WITHDRAWAL AND SCREEN 
FAILURE 
5.1 Subject Completion 
▪ 
A subject whose target ulcer has closed will be considered as having completed the study 
▪ 
A subject who completes the Treatment Phase but still has an unhealed target ulcer will be 
considered as having completed the study 
▪ 
A subject who does not complete the Treatment Phase and still has an unhealed target 
ulcer will be considered as not completing the study 
5.2 Success and Failure 
▪ 
A subject whose ulcer heals sooner than 5 weeks with periodic assessment on a twice 
weekly/weekly basis will be considered a treatment success.  The relative success of 
healing in percentage will be measured for each subject using the following calculation: 
Percentage of Healing = (Actual days to heal / 28) X 100 
▪ 
A subject whose ulcer has failed to heal by 5 weeks will be considered a treatment failure 
▪ 
A subject who experiences an amputation of the affected ulcer area is also considered a 
treatment failure 
5.3 Premature Withdrawal from the Study 
A subject who is randomized into the Treatment Period of the study but who does not 
complete the study has prematurely discontinued. 
All subjects have the right to withdraw at any point during treatment without prejudice. It will be 
documented whether or not each subject completes the study. If for any subject, study 
treatment or observations were discontinued, the reason(s) will be recorded. 
The Investigator can discontinue a subject at any time if it is considered medically necessary. 
In addition, the subject will be withdrawn from the study, if any of the following events occur: 
▪ 
A subject is significantly non-compliant with the requirements of the protocol 
▪ 
A subject becomes pregnant or does not confirm use of birth control. (Note: the pregnancy 
will be followed up to term for safety. Relevant safety information collected after the study 
has been completed will be reported as supplemental information.) 
▪ 
A subject has revascularization surgery on the lower extremity on which the target ulcer is 
located 
▪ 
The subject’s target ulcer deteriorates and is infected to the point where there is exposed 
bone 
▪ 
An infection episode lasting more than two weeks with no response to allowable 
treatments 
Premature withdrawal from the study may occur if: 
▪ 
A subject is treated with a prohibited medication. This decision will be made by the 
Investigator as to whether premature withdrawal is warranted. 
Every attempt should be made to collect follow-up information. The reason for treatment 
discontinuation or withdrawal from the study will be recorded in the source documents and on 
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the appropriate CRF. 
Before a subject is identified as lost-to-follow up, the site should make all reasonable efforts 
to contact the subject. Three follow-up attempts are required. These attempts must be 
documented and should include two phone calls and one certified letter. 
In the event that a subject is prematurely discontinued from the study at any time due to an 
adverse effect or adverse event, the procedures in Section 10 of this protocol must be 
followed. 
5.4 Screen Failures 
A subject who has signed a consent form, has been assigned a screening number, but is not 
entered into the treatment phase of the study is classified as a screen failure. Subject 
number, demography and reason for screen failure will be collected. Subjects who fail their 
first screening attempt may be re- screened again (i.e., up to two screenings total) and may 
be enrolled if they are found to meet all inclusion and none of the exclusion criteria. 
Rescreening follows a subject, not a specific wound, so subjects cannot be screened > 2 
times for different wounds. Anytime a subject screen fails and later rescreens, they must be 
re-consented for the study. 
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6 
STUDY TREATMENT 
6.1  Method for Assigning Eligible Subjects to Treatment 
Eligible subjects will be assigned to one of the two treatment groups based on a 
randomization schedule.  Details of the randomization are provided int the statistical analysis 
section. 
6.2  Helicoll®  Product Information (provided from IFU)  
 
INSTRUCTIONS FOR USE 
Helicoll® 
Semi-Occlusive, Self-Adhering and Sterilized Type-I Collagen Sheet for Wound 
Treatments, Second Degree Burns, and Chronic Ulcers. 
 
Description Of The Device 
Helicoll® is a translucent, off-white, semi-occlusive, self-adhering and pre-sterilized 
Type-I Collagen Sheet for use as a bioactive membrane. Helicoll® is flexible with 
moderate tackiness. Helicoll® is a reconstituted collagen sheet free of contaminants 
like lipids, elastin and other immunogenic proteins (US Patented). Helicoll® 
maintains a physiologically moist microenvironment at the wound surface. 
 
Intended Uses 
Helicoll® is intended for the topical wound management that includes: 
• 
Partial and full-thickness wounds. 
• 
Pressure ulcers. 
• 
Venous ulcers. 
• 
Chronic vascular ulcers. 
• 
Diabetic ulcers. 
• 
Trauma wounds (abrasions, lacerations, second-degree burns, skin tears). 
• 
Surgical wounds (donor sites/grafts, post-Mohs’ surgery, post-laser surgery, 
podiatric, wound dehiscence). 
 
Advantages Of Helicoll® Membrane 
• 
High purity type-I Collagen: Helicoll® is a patented reconstituted bioactive collagen 
sheet, free of immunogenic proteins, lipids, and elastin. 
• 
Faster Healing: Collagen phosphorylation attracts cells, regenerates tissue, and 
stimulates blood capillaries/granulation within 4 to 5 days. 
• 
Innovative Technology: Better than intact tissue-based membranes like amnion, 
intestinal wall, urinary bladder, etc. which contain 15% elastin. 
• 
Easy Application: No washing needed prior to use. 
• 
Pain Control: Effectively reduces pain. 
• 
Various Sizes: Choose from standard or customized dimensions. 
• 
Cost-Effective: Accelerated wound healing and tissue remodeling with minimal 
applications. 
• 
Long Shelf Life: Remains clinically usable for 3 years when stored in room temperature 
conditions. 
 
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Directions For Use 
Helicoll® comes in a sterile double packaging as a transparent pliable sheet. It has a back 
and a top protection cover sheet of medical grade synthetic polymer. 
 
Upon opening the sterile package, the top sheet of polymer can be removed carefully 
and the remaining can be soaked in sterile water or normal saline solution for 5 to 10 
minutes to easily remove the backing sheet. 
 
Prepare wound area using standard methods to ensure wound is free of debris and 
necrotic tissue. An initial surgical debridement of the wound may be necessary to ensure 
the wound edges contain viable tissue. 
 
Do not apply ointment or any greasy cream on the site prior to Helicoll®. Do not try to 
over stretch the membrane. 
 
Helicoll® can be applied on either of its surfaces and it adheres to the wound instantly. In 
case of dry wounds, sprinkle sterile saline solution on the surface and apply 
If there is a need to retain the skin-substitute in place, the perimetry can be taped, sutured or 
stapled as preferred by the doctor. If a secondary dressing is required, a non-adherent gauze 
with or without antibiotic can be placed to prevent unwanted adherence of the bandage to 
Helicoll®. 
 
Repeated application on every 2nd or 3rd day like a typical wound dressing is not required, 
unless the wound is infected or accumulates excessive exudate underneath, which can be 
drained by making slit openings in the Helicoll® product. 
 
Depending on the treatment modality, sometimes Helicoll® may remain intact and gets peeled 
off as the wound heals, which may carefully be removed by moistening with saline soaked 
gauze for a few minutes. However, in some cases, Helicoll® may get incorporated into the 
wound bed in about 4 to 5 days resulting in complete absorption of Helicoll®. 
 
For donor site application, after surgical removal of donor tissue, arrest bleeding by 
conventional methods, clean the site and apply Helicoll®. 
 
Oral or systemic antibiotics may be given as prescribed in infected cases and in non-infected 
cases as a preventive measure for better and faster results. 
 
CAUTION: Always handle Helicoll® using aseptic techniques. Helicoll® should not be applied 
until excessive exudate, bleeding, acute swelling, and infection is controlled. If air pockets 
appear beneath the applied Helicoll®, it can be gently pressed and removed using sterile 
methods. In case of localized bulging due to fluid accumulation beneath Helicoll®, a small 
incision can be made to exude fluid. This incision can be patched with a small piece of 
Helicoll® adhering to the original applied Helicoll® sheet. After application, use an appropriate, 
non-adherent, secondary dressing to maintain a moist wound environment. Frequency of 
secondary dressing change will depend on the volume of exudate produced and the type of 
dressing used. Do not forcibly remove sections of Helicoll® that may adhere to the wound. 
Helicoll® may form a caramel-colored gel, which can be rinsed away with gentle irrigation. 
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CONTRA-INDICATIONS: Helicoll® is derived from a bovine or ovine source and should 
not be used in patients with known sensitivity to such material. This device is not indicated 
for use in third degree burns. 
 
PRECAUTIONS: Helicoll® is sterile if the package is dry, unopened, and undamaged. Do 
not use if the package seal is broken. The device must be used prior to the expiration date. 
Discard all open and unused portions of Helicoll®. 
 
Do not re-sterilize the products and this device is intended for one time use only. 
Helicoll® is available by medical prescription only. 
 
STORAGE: Helicoll® should be stored in a clean, dry location at room 
temperature under normal storage conditions, Do Not Store Above 32℃ (90℉). 
Helicoll® has a minimum of 3 years shelf-life. 
 
STERILIZATION:  
Helicoll® has been sterilized with ethylene oxide. 
 
AVAILABLE SIZES (inches & centimeters): 
0.5 in dia disc 
(1.27 cm dia disc) 
1 sq cm 
1.0 in dia disc 
(2.54 cm dia disc) 
5 sq cm 
0.8 in x 1.6 in 
(2 cm x 4 cm) 
8 sq cm 
1.2 in x 1.6 in 
(3 cm x 4 cm) 
12 sq cm 
1.6 in x 1.6 in 
(4 cm x 4 cm) 
16 sq cm 
2 in x 2 in 
(5 cm x 5 cm) 
25 sq cm 
2 in x 4 in 
(5 cm x 10 cm) 
50 sq cm 
other custom sizes available. (Each individually 
sterile packaged) 
 
Manufactured and Marketed by: 
ENCOLL Corp. 
4576 Enterprise St., 
Fremont, CA 94538, USA 
Tel. 510-659-1466 Fx. 510-795-7571 
     website:www.helicoll.com  
     e-mail: info@encoll.com 
 
6.3  Grafix® Product Information  
 
Construction 
GRAFIX* cryopreserved placental membrane is supplied frozen in sheet form and packaged in a sterile 
cryobag contained within a heat-sealed pouch contained within a tertiary box. This packaging 
configuration allows for the introduction of GRAFIX* into the sterile field. GRAFIX PL* is supplied in 
sheet form between two (2) mesh applicators and packaged within a heat-sealed pouch contained 
within a tertiary box. This packaging configuration allows for the introduction of GRAFIX PL* into the 
sterile field. 
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Mode of Use/Application 
GRAFIX* Membrane Products can be applied in an office, hospital outpatient setting or in an operating 
room. GRAFIX* Membrane Products naturally conform to complex anatomies and may be used over 
exposed bone, tendon, joint capsule, muscle, and hardware. GRAFIX* Membrane Products may be 
used to repair acute and chronic wounds, encompassing both upper extremity and lower extremity, 
including but not limited to: diabetic foot ulcers, venous leg ulcers, pressure injuries, dehisced surgical 
wounds, burns, acute surgical wounds, pyoderma gangrenosum, and epidermolysis bullosa. 
Removal & Change Frequency 
Reapply weekly at the discretion of the responsible physician or health care professional for the 
duration of treatment. 
Additional Recommended Dressings 
Non-adherent dressing and outer dressings. 
Benefits 
• Can be used for acute and chronic wounds such as, diabetic ulcers, pressure injuries, surgical 
wounds, burns, and venous ulcers 
• Flexible, conforming cover that naturally adheres to complex anatomies and may be used over 
exposed structures such as bone, tendon, joint capsule, muscle, and hardware 
• Designed for application directly to acute and chronic wounds 
• Immune neutral 
• No need for sutures or Steri-Strips® 
• Available in multiple sizes to reduce wastage 
 
Recommended Use 
Acute Wounds 
Arterial Ulcers 
Burns 
Chronic Wounds 
Deep Wounds 
Dehisced Wounds 
Diabetic Foot 
Granulating/Epithelializing Wounds 
Pressure Ulcers 
Pyoderma Gangrenosum 
Superficial Wounds 
Surgical Dehiscence 
Surgical Incisions 
Trauma 
Venous Leg Ulcers 
Contraindications 
There are no known contraindications for these products 
. 
Warnings and Precautions 
Intended for use in one patient, on a single occasion only. 
Do not use if package integrity has been compromised. Once the user breaks the seal on the pouch, 
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GRAFIX* Membrane Products must be transplanted or discarded. 
GRAFIX* Membrane Products may not be sterilized. 
GRAFIX* Membrane Products are intended for use by qualified health care specialists such as 
physicians, podiatrists, or other appropriate health care professionals. 
The same medical/surgical conditions or complications that apply to any medical/surgical procedure 
may occur during or following application. 
The health care professional is responsible for informing the patient of the risks associated with his/her 
treatment and the possibility of complications or adverse reactions. 
Caution should be exercised for patients with known sensitivities for reagents used for processing, 
disinfection, and storage which may remain on the product. 
 
Adverse Effects/Reactions 
Donor screening methods are limited; therefore, certain diseases may not be detected. The following 
complications of tissue transplantation may occur: 
• Transmission of infectious agents or diseases of known or unknown etiology including, but not limited 
to fungi, bacteria, or viruses; 
• Immune rejection of implanted GRAFIX* Membrane Products; or 
• Loss of function and/or integrity of GRAFIX* Membrane Products due to resorption, fragmentation, 
and/or disintegration. 
 
Storage Requirements 
GRAFIX* Cryopreserved Placental Membrane has a 3 year shelf life and and should be stored frozen 
at -75°C to -85°C (-103°F to -121°F). GRAFIX* PL Lyopreserved Placental Membrane Tissue has a 2 
year shelf life and should be stored at room temperature. 
 
How Supplied/Sizing 
1.5cmx2cm, 2cmx3cm, 3cmx4cm, 5cmx5cm. Round: 16mm. 
 
6.4  Epifix® Product Information  
 
Description 
Human amniotic membrane is a thin, collagenous membrane derived from the 
placenta, the area in which the human fetus grows and develops within the mother’s 
uterus. Human amniotic membrane consists of multiple layers.  
EpiFix is a minimally manipulated, dehydrated, non-viable cellular amniotic membrane 
allograft that contains multiple extracellular matrix proteins, growth factors, cytokines 
and other specialty proteins present in amniotic tissue to provide a barrier membrane 
that enhances healing.  
EpiFix allografts are human tissue products and appearance may vary between 
donors. Variations in color (tan to light brown), opacity, and thickness are normal due 
to the nature of human tissue. 
 
Tissue Uses  
EpiFix Amniotic Membrane Allograft is intended for homologous use in the treatment of acute and 
chronic wounds to reduce scar tissue formation, modulate inflammation, provide a barrier, and 
enhance healing.  
 
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Contraindications  
EpiFix should not be used on (1) areas with active or latent infection and/or (2) a patient with a disorder 
that would create an unacceptable risk of post-operative complications.  
 
Precautions/Warnings  
• EpiFix allografts remain suitable for transplantation in an unopened, undamaged package, under 
proper storage conditions.  
• Please inspect the integrity of the package upon receipt. If package and contents appear defective or 
damaged in any way, immediately contact the distributor.  
• This allograft is intended for single-patient use only. Discard all unused material.  
• The procedure should be performed by an authorized medical professional.  
• Strict donor screening and laboratory testing, along with dedicated processing and sterilization 
methods, are employed to reduce the risk of any disease transmission. However, as with all biological 
implants, an absolute guarantee of tissue safety is not possible. This allograft has the potential to 
transmit infectious disease to the recipient.  
• The reaction of the body to any biological implant is not completely understood.  
• Caution should be used when treating patients with a known sensitivity to aminoglycoside antibiotics. 
• Discard all damaged, mishandled or potentially contaminated tissue.  
• This product has not been tested in combination with other products.  
• DO NOT RE-STERILIZE. 
 
Preparation, Reconstitution and Use  
Prior to implantation, carefully follow the EpiFix allograft preparation steps below using aseptic 
technique:  
 
Wound Bed Preparation  
• Ensure the wound is free from clinical signs of infection.  
• Prepare wound bed as needed.  
 
Removing EpiFix from Packaging  
• The outer peel pouch is NOT sterile. The inner pouch that contains EpiFix is sterile (unless the 
pouches are damaged or compromised). 
 • Carefully open the peelable corner of the outer pouch and extract the inner pouch using aseptic 
technique. Ensure the inner pouch does not come in contact with any portions of non-sterile surface of 
the outer pouch.  
• Using aseptic technique, SLOWLY peel a corner of the inner peel pouch and grasp EpiFix with 
fingers or non-toothed, sterile forceps.  
• Use EpiFix promptly after opening the inner, sterile pouch.  
 
PLEASE TAKE GREAT CARE WHEN REMOVING EpiFix FROM THE INTERNAL POUCH. EpiFix 
IS THIN AND EXTREMELY LIGHTWEIGHT.  
 
EpiFix Preparation  
1. In a dry state, use sterile dry scissors to cut EpiFix to fit within the wound margins. It is acceptable to 
overlap the wound margins with EpiFix by 1mm.  
2. EpiFix can be applied wet or dry.  
3. EpiFix can be hydrated while on the wound site with sterile saline solution. Simply apply several 
drops of sterile solution to EpiFix. During and following hydration, the embossment on EpiFix will begin 
to fade. 
 
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EpiFix Orientation & Application  
EpiFix should be placed on the wound site, using the orientation of the embossment lettering as a 
guide. Proper orientation of EpiFix can be noted when the embossment nomenclature reads correctly 
from left to right. If the mesh process has diminished the ability to clearly read the embossment, the 
following two alternate methods may be used for placement:  
1. An elongated, horizontal perforation is located in the top left area of the graft connecting two 
adjacent perforations. Proper orientation can be noted when the graft is placed on the wound site 
such that this horizontal perforation remains visually in the top left area of the graft.  
2. The clear side of the inner pouch is indicative of the UP side of the graft orientation. Proper 
placement can be achieved by removing the graft from the pouch with the clear side up and 
moving to the wound site with that same orientation.  
Absorbable, non-absorbable suture material and/or tissue adhesives can be used to fixate EpiFix to the 
wound site, if desired.  
 
Primary Dressing  
• EpiFix should be covered with a non-adherent contact layer.  
• EpiFix should NOT be disturbed, if possible, for several days or before the next application, if needed. 
• If an infection occurs at the graft site, treat infection per institution’s protocol. Secondary Dressing  
• EpiFix requires a moist wound environment. Use appropriate moisture management dressings for the 
wound type and treatment ideology.  
 
Support Therapies  
• EpiFix is compatible with offloading/compression/negative pressure therapies.  
• EpiFix can be used in conjunction with hyperbaric oxygen therapy.  
 
Re-application of EpiFix  
• It is recommended that EpiFix grafts are applied weekly until wound epithelialization is achieved. 
However, clinician discretion should be used based on patient and wound condition/progress. It is 
clinically acceptable to apply EpiFix on a biweekly basis if desired.  
 
Adverse Effects & Reporting  
• As with any procedure, the possibility of infection exists.  
• Proprietary processing and validated sterilization methods are employed to eliminate potential 
deleterious components of the allograft. However, as with all biological implants, the possibility of 
rejection exists.  
• Any adverse reactions, including the suspected transmission of disease attributable to this allograft, 
should be reported immediately to MiMedx®.  
 
Acceptable Storage  
EpiFix allografts should be stored in a clean, dry environment at ambient conditions. EpiFix allografts 
have a 5 year shelf life. Check the label for the expiration date. 
 
 
 
 
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7 
STANDARD OF CARE, CONCOMITANT MEDICATIONS, 
EXCLUDED/ALLOWED THERAPIES/MEDICATIONS AND ALLOWED 
DRESSINGS 
Beginning at the screening visit, ALL subjects must have their target ulcer managed using the 
SOC procedures noted below. 
7.1 SOC Procedures 
7.1.1 Cleaning the Target Ulcer 
Remove all dressings. The leg should be elevated for as much time as possible during this 
process. Wash the foot with sterile water or saline solutions. Gently irrigate the target ulcer 
prior to each dressing change with the same solution. Wound cleansers containing antiseptics 
(examples: Hypochlorous Acid, Vashe, ExSept, PrepIt, etc.) are prohibited for use during the 
duration of this study. 
During the Treatment Phase, the washing and cleaning of the DFU must be done prior to 
application of SOC primary dressing. 
 
For the TV2 and TV3 additional visit where application of a new Helicoll® does not occur, the 
primary dressing will be lifted from the wound site along with the Helicoll® Advanced Skin 
Substitute or Epifix® or Grafix®.  If the Helicoll® Advanced Skin Substitute is adherent to the 
wound, moisten gently with saline before attempting to remove. Cleaning of the target ulcer 
as noted above is suggested, however debridement should be limited, and it is suggested that 
only a very gentle removal of any slough be performed at the investigator discretion. Digital 
Photography is then taken and the Helicoll® Advanced Skin Substitute or Epifix® or Grafix® 
and primary dressing should be placed back on the wound. If the primary dressing is soiled or 
damaged a new primary dressing can be applied.  
7.1.2 Debridement of the Target Ulcer 
Debridement is an essential technique and SOC in the treatment of DFU. It is important to 
remove all non-viable and necrotic material from the target ulcer prior to enrolling the patient. 
Debridement is allowed during the treatment phase at the treating Investigator’s discretion. 
For detailed guidelines please see Appendix E as well as recommendations for debridement 
during the 2nd visit in week 2 and 3 in which a new Helicoll® is not placed. 
7.1.3 Offloading 
Offloading is essential if the wound is to heal. A diabetic offloading CAM boot will be given to 
each subject, and this may be augmented with felt or foam to offload the ulcer or if the 
subject cannot be accommodated into a CAM boot, a TCC will be provided. 
Subjects will be educated on the importance of using the device to offload their DFU and 
instructed on keeping dressings dry and to call or visit the study site if the dressing becomes 
soiled, wet or is removed. In addition, subjects should be educated on wound infection and if 
they observe infection, to call or visit the study site. 
7.2 Concomitant Medications 
The subject may be administered any necessary medications, at the discretion of the 
Investigator; provided such medications are not applied topically to the target ulcer surface 
(topical medication can be applied to other surfaces around the ulcer or other non-reference 
ulcers). 
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All medications and therapies administered or taken by the subject beginning 30 days prior to 
signing the ICF and throughout the study will be recorded in the source documents and for 
randomized subjects, on the appropriate CRF. 
7.3 Prohibited Medications and Therapies 
The following treatments and medications are prohibited 30-days prior to first screening visit and 
throughout the trial: 
▪ 
Any cellular and/or tissue-based products or wound dressings that include growth factors 
(e.g., Regranex, Dermagraft, Apligraf, GraftJacket, OASIS, Primatrix, Matristem) placed 
on the target ulcer 
▪ 
Revascularization surgery on the lower extremity with the target ulcer  
▪ 
Radiation therapy to the foot 
7.3.1 Excluded starting at SV1 
▪ 
Topical antibiotics* or other topical agents, including or containing silver, honey, hydrofera 
blue, etc. (with the exception of anesthetics used during debridement) 
▪ 
Systemic steroids/oral corticosteroids in excess of 10mg daily dose. (Note: inhaled 
steroids are acceptable) 
▪ 
Other immunosuppressive agents and chemotherapy 
▪ 
Cytotoxic therapies 
▪ 
Negative pressure wound therapies 
▪ 
Hyperbaric oxygen 
▪ 
Any other investigational treatment/medications in the Investigator’s opinion are likely to 
seriously impact wound healing 
▪ 
Heat lamps 
▪ 
UV lights 
▪ 
Whirlpool baths 
▪ 
Water Piks 
▪ 
Jet water streams (other than gentle water irrigation) 
▪ 
Wound cleansers that contain active ingredients and are not just normal saline (e.g., 
Hypochlorous Acid, Vashe, ExSept, PrepIt, etc.) 
▪ 
Selective COX-2 inhibitors (such as Celecoxib) 
*Topical antibiotics must be stopped at the first screening visit. 
7.4 Allowable Medications and Therapies 
The following medications and therapies are allowed during the study, if in the opinion of the 
Investigator, they are required for proper care of the study subject: 
▪ 
Use of anesthetics for debridement 
▪ 
Treatment with systemic antibiotics for acute or chronic infection, however, prophylactic 
use of systemic antibiotics is not allowed 
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▪ 
Other medications/therapies that are not otherwise prohibited and in the opinion of the 
Investigator, are required for proper medical care 
7.5 Approved Dressings 
The following is a list of approved dressings suitable for study DFUs receiving SOC 
treatment: 
SOC Helicoll® Group 
▪ 
Helicoll® – Advanced Skin Substitute  
Helicoll Sizes intended for this study 
Grafts per box 
0.8 in x 1.6 in 
2 cm x 4 cm 
(8 sq cm) 
2 
1.6 in x 1.6 in 
4 cm x 4 cm 
(16 sq cm) 
2 
 
▪ 
Plain foam (e.g., Allevyn gentle, Mepilex) or Mepitel/Adaptic Touch 
▪ 
Three-layer dressing of first layer - non-adherent and porous, second layer – absorbent 4x4 gauze 
pads & third layer - soft cast roll and ace bandage/Coban 
SOC Epifix® or Grafix® Group 
▪ 
Epifix® or Grafix®  
▪ 
Plain foam (e.g., Allevyn gentle, Mepilex) or Mepitel/Adaptic Touch 
▪ 
Three-layer dressing of first layer - non-adherent and porous, second layer – absorbent 4x4 gauze 
pads & third layer - soft cast roll and ace bandage/Coban 
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8 DESCRIPTION OF PROTOCOL ASSESSMENTS AND PROCEDURES 
8.1 Informed Consent 
Written informed consent will be obtained for this study by the Investigator or suitably qualified 
designee from all subjects before the collection of any protected health information and 
performance of any protocol-specific procedure. 
In obtaining and documenting informed consent, the Investigator must comply with applicable 
regulatory requirements and must adhere to Good Clinical Practice (GCP). The Investigator, 
or designee, must fully inform subjects of all pertinent aspects of the study. Before informed 
consent may be obtained, the Investigator, or a person designated by the Investigator, must 
provide the subject ample time and opportunity to inquire about details of the study and to 
decide whether or not to participate in the study. All questions about the study must be 
answered to the satisfaction of the subject. Prior to the subject’s participation in the study, the 
ICF must be signed and personally dated by the subject and by the person who conducted the 
informed consent discussion. For screen failure subjects who rescreen at a later date, re- 
consenting is required. 
8.2 Assessment of Eligibility 
During the Screening Visit/Period, the Investigator must assess a subject’s continued 
suitability and eligibility for the study, especially with regard to the Inclusion and Exclusion 
criteria. If the subject is no longer suitable or eligible for the study, the subject will be 
considered a screen failure. Screen failure subjects may be re-entered into the study at a later 
time and re-screened. 
8.3 Subject Demographics, Medical History and Ulcer History 
8.3.1 Demographics 
For the purposes of this study, demographic information will include: 
▪ 
Date of ICF signature 
▪ 
Date of birth 
▪ 
Gender at birth 
▪ 
Race (American Indian/Alaskan Native, Asian, Black/African American, Native 
Hawaiian/Pacific Islander, Caucasian or other) 
▪ 
Ethnicity (Hispanic/Latino or Not Hispanic/Latino) 
▪ 
Use of tobacco products 
8.3.2 Medical History 
A medical history will be recorded during the Screening Period and will include: 
▪ 
All ongoing medical conditions 
▪ 
All previously resolved medical conditions related to diabetes or foot ulceration or which 
are relevant in the opinion of the Investigator 
Events that emerge prior to the randomization visit will be recorded in the medical history and 
not as Adverse Events. Aside from being used to determine subject eligibility, this information 
will permit the Investigator to record the nature, duration and severity of any ongoing baseline 
medical conditions that occur prior to randomization.
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Medical histories will be recorded using the body system categories outlined below: 
▪ 
Cardiovascular 
▪ 
Lymphatic 
▪ 
Respiratory 
▪ 
Hematologic 
▪ 
Gastrointestinal 
▪ 
Immunologic 
▪ 
Renal 
▪ 
Dermatologic 
▪ 
Hepatic 
▪ 
Psychiatric 
▪ 
Neurological 
▪ 
Genitourinary 
▪ 
Endocrine 
▪ 
Other 
▪ 
DFU History 
For each relevant history, the following will be documented: 
▪ 
Disease/disorder/condition 
▪ 
Date of diagnosis 
▪ 
History status (resolved or ongoing) 
8.3.3 Foot Ulcer History 
▪ 
Duration of the current DFU 
Note: “Duration” is defined as the length of time that the target ulcer has been open at this 
location since the last time it was fully closed. 
▪ 
Current offloading system used for the DFU (if any) and length of time that this has been 
used 
▪ 
Prior treatments that have been used on the DFU for up to 1 year 
▪ 
Age when the subject developed his/her first DFU 
▪ 
Total number of previous DFUs 
▪ 
Location of the current DFU 
Note: “Target ulcer location” is defined by the ulcer being on the left or right foot, by the 
location of the ulcer on the foot, dorsal or plantar, toe (which toe), forefoot, midfoot, hind 
foot, ankle heel and by the positioning of the ulcer as lateral, medial, dorsal, or plantar 
located. 
▪ 
Number of additional DFUs and location of each present at the screening visits 
▪ 
History of DFU recurrence 
Note: “Recurrence” is defined as the re-opening of the target ulcer after complete healing. 
▪ 
History of any amputations in the study foot and contralateral foot or leg 
▪ 
History of any significant foot deformities, dermatological abnormalities, fungal lesions, or 
other findings. 
8.3.4 Physical Exam 
The physical examination will include routine examinations for the following: 
▪ 
Head, ears, eyes, nose, and throat 
▪ 
Abnormalities of the extremities 
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▪ 
Neurologic abnormalities 
▪ 
Heart/cardiovascular abnormalities 
▪ 
Musculoskeletal abnormalities 
▪ 
Dermatologic abnormalities 
▪ 
Any other body system for which an abnormality is noted and which, in the opinion of the 
Investigator, is relevant to the safety of the subject or could impact safety or efficacy 
results for the subject, i.e., the abnormality is clinically significant 
Each abnormality will be recorded, and the Investigator will record an assessment of its 
clinical significance. 
8.3.5 Vital Signs 
The following vital signs will be collected: 
▪ 
Height 
▪ 
Weight 
▪ 
Seated blood pressure (take after the subject has been seated for at least 5 minutes) 
▪ 
Pulse 
▪ 
Temperature 
8.3.6 
Pregnancy test 
Females who are of childbearing potential must have a urine or blood pregnancy test at SV1. 
8.3.7 
Laboratory tests 
The following laboratory values will be collected per the schedule of visits (Table 1): 
▪ 
Serum Creatinine 
▪ 
HbA1c 
▪ 
Blood Glucose 
8.4 
Peripheral Perfusion Assessment 
One of the following assessments will be completed to confirm sufficient tissue perfusion for 
trial participation. 
8.4.1 ABI or Arterial Doppler Ultrasound 
ABI, also known as the Winsor Index and the Ankle Brachial Pressure Index, is the ratio of 
blood pressure measured at the ankle to that measured at the arm. Details of the procedure 
can be found in Appendix B. An ABI < 0.8 indicates that there is a high probability that arterial 
insufficiency is present (positive predictive value 95% in a general practice population). 
It should be noted that incompressible, calcified arteries may occur in diabetes causing a 
falsely elevated ABI, so if the subject has other signs or symptoms that could suggest 
peripheral arterial disease, further investigations to determine vascular status may be 
warranted. 
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A documented record of an ABI test performed using the target ulcer leg, within one month of 
SV1 is acceptable for the purposes of this study, otherwise this must be completed in the 
Screening Period. 
As an alternative, arterial Doppler ultrasound can be performed evaluating for biphasic dorsalis 
pedis and posterior tibial vessels at the level of the ankle. 
ABI test to be included in week 1, 1.5 and 2 to monitor neo-vascularization. 
8.4.2 SPP 
SPP provides another noninvasive method for measuring microcirculatory pressure of the 
artery at the skin level. It measures the pressure at which perfusion first returns to the 
cutaneous microcirculation following a controlled release of occlusion. SPP is useful in 
assessing the ischemic severity of lower limb and predicting ulcer healing in chronic critical 
limb ischemia with a value of <30 mmHg. See Appendix C for the appropriate technique of 
assessment. 
8.4.3 TCOM 
A TCOM assesses the partial pressure of oxygen molecules dissolved in the blood plasma. 
The amount of oxygen detected by the sensor is a balance of the oxygen delivery and local 
physiologic demands and reflects the metabolic status of the skin; it indicates the level of 
oxygen available at the ulcer site to assist in the ulcer healing process. A TCOM less than 30 
mmHg of oxygen indicates impaired ulcer healing and ischemic disease. An appropriate 
technique for a TCOM assessment is provided in Appendix D. 
8.5 
Pain Assessment: NPRS 
Pain intensity of the reference DFU is to be assessed before any dressing changes or other 
ulcer manipulations at all screening and treatment visits. 
The subject will be asked to indicate a numerical value that best represents the current pain 
intensity at the target ulcer site on a scale of 0 to 10, anchored by word descriptors at each 
end, as "no pain" on the left side and "worst possible pain" on the right side of the number line. 
The number 0 represents “no pain,” the number 5 represents “moderate pain”, and the 
number 10 represents the “worst possible pain.” The subject indicates the level of pain 
intensity by selecting a number on the line shown in Figure 3 below that represents their 
perception of their current state. 
 
 
FIGURE 3: SUBJECT PAIN SCALE (NPRS) 
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8.6 
Ulcer Assessments 
A number of different target ulcer assessments are required, including appearance, exudate 
characteristics, infection, and peri-wound characteristics. 
8.6.1 Target Ulcer Appearance Assessment (Pre-debridement) 
Target Ulcer Appearance Assessment (pre-debridement) categories includes: 
▪ 
% Granulating (red/brown bumpy tissue that bleeds upon light debridement) 
▪ 
% Non-viable tissue 
▪ 
% Epithelialized Skin 
Note: the categories should add to 100%. 
8.6.2 
Target Ulcer Exudate Assessments 
The Investigator will determine the amount and type, if any, of target ulcer exudate. In 
determining the amount of target ulcer exudate, the Investigator must take into account the 
amount of exudate absorbed into the target ulcer dressing. Table 2 below details the 
categories will be used to quantify the amount and describe the type of target ulcer exudate: 
TABLE 2: TARGET ULCER EXUDATE ASSESSMENT 
 
Target Ulcer Exudate Assessment 
Volume 
• 
No exudate 
• 
Minimal amount 
• 
Light (scant) or small amount 
• 
Moderate amount 
• 
Heavy/large/copious amounts 
Type 
• 
Not applicable: no exudate present 
• 
Serous: clear or light-yellow watery plasma 
• 
Serosanguinous: pink to light-red watery plasma 
• 
Sanguineous: red with fresh bleeding 
• 
Purulent: thick and opaque exudate, of creamy yellow, green, white, 
or tan color 
 
8.6.3 Target Ulcer Infection Assessments 
The presence/absence of the following signs of infection at the target ulcer site will be 
documented at each visit. Infection of the target ulcer will be assessed using the STONEES 
method developed by Woo and Sibbald.19 Table 3 below lists the criteria for considering an 
target ulcer infected: 
TABLE 3: TARGET ULCER INFECTION ASSESSMENT 
 
Target Ulcer Infection Assessment 
Three or more of the following signs or symptoms are present: 
• 
Increased surface area 
• 
Increased peri-wound margin temperature by more than 3⁰F difference 
between two mirror image sites 
• 
Exposed bone or can be probed to the bone 
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Infection at Target Ulcer site prior to Randomization  
If the infection occurs prior to randomization i.e., prior to TV1, then the subject will be ineligible 
to be randomized. 
Infection at Target Ulcer site after Randomization  
If infection of the target ulcer site occurs after randomization i.e., after Randomization visit 
record the infection as an adverse event and treat it as appropriate with oral antibiotics at the 
discretion of the Investigator. 
 
Note: application of topical antibiotics and anti-microbial dressings to the target ulcer site are 
prohibited. 
A subject with an infected target ulcer that is being treated by the Investigator will remain in the 
study unless the situation requires an alternative methodology that violates the protocol. 
Antibiotic interventions will be recorded on the Concomitant Medications CRF, and the event 
will be categorized as an Adverse Event, serious if it meets the definition of that category. All 
subjects who show evidence of an ulcer infection must have it reported on an Adverse Event 
CRF. 
All subjects will be instructed to contact the Investigator if signs or symptoms of infection 
develop prior to their next scheduled visit. 
During an episode of infection, the Investigator should not continue TCC, if it is being used, 
until the infection is resolved, and dressings should be changed at least every 72 hours. 
Evaluate the skin surrounding the target ulcer for presence of signs such as erythema, edema, 
and cellulitis. 
8.6.4 Investigator Assessment of Target Ulcer Closure 
“Complete healing” of the target ulcer is defined as 100% re-epithelialization without drainage. 
At each visit, the Investigator will assess the ulcer by answering the following questions: 
• 
Is target ulcer 100% re-epithelialized? 
• 
Is drainage absent? 
Both questions must be answered “yes” for the target ulcer to be considering having reached 
“complete closure.” 
The date of complete healing is defined as the date of the first assessment of 100% re- 
epithelialization. 
Target Ulcer Photographs and Measurements 
The target ulcer will be digitally imaged with a digital camera that is at least 20 (twenty) 
megapixels, with the photo taken at a focal distance of approximately 18 inches and a two-
dimensional calibration scale with two photos taken at a 
• 
New areas of breakdown or satellite lesions 
• 
Presence of swelling or reddened skin in peri-wound area 
• 
Increased wound drainage 
• 
Unpleasant, sweet, or sickening odor present 
NOTE: An ulcer that is deemed to be infected with three or more of the following 
signs will undergo a wound culture to confirm the presence of infection and identify 
the pathogenic organism, so the study subject receives the most appropriate 
antibiotic treatment 
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distance of approximately 18 inches and one taken close up in good focus. The Investigator 
will also take a ruler and measure length and width of the wound and record appropriately. 
The area will be calculated using a length by width calculation.  
8.7 
 Randomization  
Subjects who are eligible to participate in the study will be randomized to one treatment group 
at the time of completion of screening. 
8.8 
Assessment of Offloading 
The following questions will be asked to assess the performance of the offloading during the 
Screening and Treatment Phase visits and at selected follow-up visits: 
• 
Did the subject have the offloading device with him or her or is the TCC cast still present? 
(Yes/No) 
• 
What percentage of the time is the subject using the offloading device while not sleeping? 
(Note: heel ulcers might still need to be offloaded while sleeping.) 
8.9 
Semmes Weinstein Monofilament Test for Peripheral Neuropathy 
This is a non-invasive topical evaluation of neuropathy with a monofilament wire to the foot 
with the target ulcer. The test will be performed in a standard manner with 10 points being 
evaluated on each visit. At each point, the Investigator asks the subject whether they can feel 
the filament (yes or no). Note: If a subject is missing a body part called out in the Semmes 
Weinstein test, the most skin in the area where the missing body part is located is used for 
the test. 
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9 
DATA AND STATISTICAL ANALYSES 
9.1 Data Analysis 
This study consists of primary, secondary and exploratory endpoints. Data analysis of study 
endpoints includes use of validated DFU wound healing measures, quantitative and qualitative 
analyses, as well as exploratory methodologies. Table 4 below details each study endpoint, 
corresponding data, and planned analyses. 
TABLE 4: DFU ENCOLL ENDPOINT DATA ANALYSIS 
 
Endpoint 
Endpoint Description 
Supporting Study Data Endpoint Analysis 
Primary 
Percentage wound area 
reduction from TV1-TV5 
measured manually with 
digital photography 
Digital imaging 
Mathematical wound 
area change 
Secondary 
Mean number of repeated 
applications of the 
Advanced Skin Substitute 
used to obtain wound 
closure  
Data on IP application at 
each visit 
Data Management will determine 
during review/analysis 
The proportion of subjects 
that obtain complete 
closure over the 5-week 
treatment period 
Investigator assessment 
of periodic healing (up 
to 100% epithelization 
w/no drainage), 
measurements of ulcer 
size using digital photos 
Study Chair will determine using 
photographs of the DFU 
The time to achieve 
complete wound closure of 
the target ulcer by the end 
of 5 weeks 
Data on the progress of 
wound healing 
measured at different 
time periods  
Mathematical time based on 
healing 
The relative success of healing in 
percentage will be measured for 
each subject using the following 
calculation: 
Percentage of Healing = (Actual 
days to heal / 28) X 100 
Exploratory 
The appearance, structural 
stability, and fragility of the 
new skin formed 
documented at each visit. 
Any recurrence of the 
wound also will be 
monitored. 
 
Observation 
Complete listing of TEAEs, 
categorized by treatment group 
and system organ class 
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9.2 
Statistical Analysis 
Descriptive statistical methods will be used to summarize the data from this study with no 
hypothesis testing performed for the endpoints. 
A formal Statistical Analysis Plan (SAP) will be created prior to database lock. The primary 
endpoint analysis will be at 5 weeks and a series of secondary and exploratory endpoints will 
be analyzed at 5 weeks (PAR will be analyzed at all timepoints). The SAP will provide a more 
technical and detailed description of the proposed data analysis methods and procedures. Any 
deviation from the analyses outlined in the protocol will be described in the most current 
version of the SAP. All statistical analyses will be conducted using PASW 28. 
No power analysis was conducted as the numbers of subjects enrolled in this trial are too 
small.   
The populations defined for analysis will include the intent-to-treat (ITT) and per protocol (PP). 
Additional analysis populations may be defined to evaluate study results. Any additional 
analysis populations will be defined in the SAP. 
 
Primary analysis: The percentage wound area reduction from TV1 to TV5 measured manually with digital 
photography 
 
Secondary analysis: The time to achieve complete wound closure of the target ulcer by the end of 5 
weeks 
The proportion of subjects that obtain complete closure over the 5-week treatment period 
Mean number of repeated applications of the Advanced Skin Substitute used to obtain wound closure 
Endpoint analysis: The appearance, structural stability, and fragility of the new skin formed are 
documented at each visit.  Any recurrence of the wound also will be monitored.  
Safety analysis: Tabular listings of all TEAEs per treatment group will be created using system 
organ class. Other analysis will be described in the SAP. 
Every effort will be made to obtain required data at each scheduled evaluation from all 
subjects who have been enrolled and treated. Subjects who are withdrawn or lost to follow-
up will be included in the ITT analysis of primary and secondary endpoints using data 
imputation methods detailed in the SAP. Sensitivity analysis will also be conducted for the 
primary endpoint using different imputation algorithms. 
 The number and type of 
Treatment Emergent 
Adverse Events 
 
 
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10 
ADVERSE EFFECTS AND EVENTS (DEFINITIONS AND REPORTING) 
The Investigator is responsible for the detection and documentation of any events meeting the 
criteria and definition of an adverse effect or adverse event (AE), as described in this protocol. 
During the study when there is a safety evaluation, the Investigator or site staff will be 
responsible for detecting, documenting, and reporting any adverse effect or adverse event, as 
detailed in this section of the protocol. 
10.1 SOC Adverse Events 
Since this a post market study involving commercially available SOC dressings of FDA 
cleared and approved medical devices; adverse events associated with their use may also 
require reporting to FDA along with the possible reason for the failure of the treatment 
which may include product handling and other details. 
An AE is defined as any unfavorable or unintended sign, symptom or disease that occurs or is 
reported by the subject to have occurred, or a worsening of a pre-existing condition related to 
the procedure or medical treatment (device/drug) used. All AEs related to the study procedure 
or medical treatment, including intercurrent illnesses, must be recorded in the subject’s 
medical records and on the CRF. CTCAE v5.0 will be used for AE reporting. 
AEs will be defined as those events that occur after the first study treatment is applied at TV1, 
through the final study visit. A description of the AE along with the onset date, end date, 
severity, action taken, treatment, outcome, likely cause and relationship to the study 
procedure or products will be recorded in the CRF. 
AEs associated with each treatment arm will be tabulated and compared at the end of the 
study. AEs will be elicited through direct questioning, subject reports, and physical 
examination. 
An abnormal laboratory test result is not by itself considered to be an AE unless the Investigator 
considers the finding of clinical significance that should be reported in such a manner. 
The Investigator is responsible for assessing the relationship of the AE to the procedure or 
medical treatments used and the seriousness and expectedness of the AE at the time of 
occurrence. A medically qualified person appointed by the Sponsor will also assess this once 
the Sponsor has been notified of an AE. For enrolled subjects, all AEs that occur during the 
trial will be documented on the AE CRF. 
AEs reported during the study should be followed to resolution of the AE or within thirty days 
from the end of the study. A final assessment of the outcome will be made at that time. 
Each AE related to the study procedure or medical treatment used will be categorized as 
“serious” or “not serious,” based on the definition of a serious adverse event (SAE). A SAE is 
defined as an AE resulting in at least one of the outcomes described in Section 10.1.3. 
10.1.1 AE Severity Assessments 
The guidelines outlined in CTCAE v4.0 will be used for severity assessments. Note: the term 
“severe” is a measure of intensity and that a severe AE is not necessarily serious. Table 5 below 
provides guidance on determining the severity of an AE. 
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TABLE 5: AE SEVERITY GRADING SCALE 
 
Severity Grade 
Description 
Mild (1) 
Awareness of sign, symptom, or event, but easily tolerated; 
does not interfere with usual daily activities or tasks. 
Moderate (2) 
Discomfort is enough to cause interference with usual daily 
activity. It may warrant therapeutic intervention. 
Severe (3) 
Incapacitating: inability to perform usual activities and daily 
tasks; significantly affects clinical status; requires therapeutic 
intervention. 
Life-threatening (4) 
Emergency treatment required, life-threatening, death. 
A grade of 1-4 is assigned by the Investigator to each AE. 
10.1.2 AE Causality Assessments 
AEs will be assigned a relationship (causality) to the study procedure or medical treatments. 
The Investigator will be responsible for determining the relationship between an AE and the 
study procedure or medical treatment. The type of event, organ system affected and timing of 
onset of the event will be factors in assessing the likelihood that an AE is related to the study 
procedure or medical treatment. The relationship of AEs to study procedures or medical 
treatments will be classified as follows: 
▪ 
Not Related: No relationship exists between the AE and the study procedure or medical 
treatment. The event is attributed to a pre-existing medical condition or an inter-current 
event unrelated to the study procedure or medical treatments. 
▪ 
Possibly Related: Follows the study procedure or medical treatment but may have 
developed as a result of an underlying clinical condition or treatments/interventions 
unrelated to the study. 
▪ 
Probably Related: Follows the study procedure or medical treatment but is unlikely to have 
developed as a result of the subject’s underlying clinical condition or other treatment or 
other interventions. 
▪ 
Definitely Related: Follows the study procedure or medical treatment and physical 
evidence shows a convincing relationship to the study procedure or medical treatment. 
▪ 
Unknown: Follows the study product or medical treatment, but unable to determine the 
relationship to the study procedure or medical treatment. 
10.1.3 Serious Adverse Events 
A SAE is any untoward medical incident that occurs during the course of the study beginning 
after informed consent has been executed. 
A SAE is defined as any AE that: 
▪ 
Results in death 
▪ 
Is life threatening (the subject is at immediate risk of dying from the adverse experience) 
▪ 
Requires subject hospitalization or prolongs existing hospitalization 
▪ 
Results in persistent or significant disability/incapacity 
▪ 
Is a congenital anomaly/birth defect 
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Note: Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a SAE when, based upon appropriate medical judgment, 
they may jeopardize the subject and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition. 
10.1.3.1 Reporting of Serious Adverse Events 
The Investigator or delegated site personnel is required to report all SAEs that occur during 
the study. Once the Investigator or delegated site personnel becomes aware of a SAE, he/she 
must report the SAE to the Sponsor and copy the CRO within 24 hours.  
The study sponsor contact is: Subra Guna, PhD 
Phone:  510-396-8581 
Email:  guna@encoll.com 
A written SAE report must follow and must include a full description of the event and all 
supporting documentation available at the time (e.g., lab reports, culture reports, etc.). 
Additional follow-up information as it becomes available must be reported to the CRO and/or 
Sponsor. 
The Investigator or delegated site personnel is also responsible for reporting all SAEs to the 
IRB in accordance with IRB reporting guidelines, local laws and regulations. The Investigator 
is responsible for maintaining documentation in the study file that indicates the IRB has been 
properly notified. 
10.1.3.2 
SAE Follow-up 
All SAEs will be monitored for a minimum of 30 days until they are resolved, have stabilized, 
or are clearly determined to be due to a subject’s stable or chronic condition or intercurrent 
illness (es). 
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11 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTATION 
Subjects will be identified on CRFs by a unique subject identification number and on source 
documents by name and date of birth. No personal identifier will be used in any publication or 
communication used to support this research study. The subject identification number will be 
used if it becomes necessary to identify data specific to a single subject. 
The monitors, auditors, personnel authorized by the Sponsor, the local IRB and applicable 
regulatory bodies are eligible to review medical and research records related to this study as a 
part of their responsibility to protect human subjects in clinical research and will be given 
direct access to source data and documentation (e.g., medical charts/records, printouts, etc.) 
for source data verification, provided that subject confidentiality is maintained in accordance 
with local requirements. Access to electronic medical records may be governed by institution 
policy and each site will be required to ensure access while remaining compliant with 
institutional requirements. 
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12 
QUALITY CONTROL AND QUALITY ASSURANCE 
12.1 
Acceptability of CRF 
CRFs must be completed for each subject who has signed an ICF. For subjects who are 
screen failures, this would be limited to Screening or other applicable forms. All source 
documents and CRFs will be completed as soon as possible after the subject’s visit. 
12.2 
Modification of Protocol 
The Investigator will not modify or alter this protocol without first obtaining the concurrence of 
the Sponsor. Approval by the Investigator’s IRB must also be obtained prior to implementation 
of the change, with two exceptions: 
▪ 
When necessary to eliminate apparent immediate hazard to the subject, or 
▪ 
When the modification does not involve the subject’s participation in the trial. 
An amendment may also require modification of the ICF. The Investigator will provide an 
approval letter for the amendment and revised ICF, if applicable, to the Sponsor. An 
amendment must be made in writing, and it must be dated by both the Sponsor and the 
Investigator. All material must be approved by the IRB. 
12.3 
Reporting Protocol Deviations 
The Investigator is obligated to follow the protocol without departure from the requirements 
written in the protocol. Any time there is deviation from any protocol requirements, a protocol 
deviation is required to be documented within the subject’s source documentation. If the 
deviation may impact subject participation, the Sponsor should be notified and will make the 
determination as to whether the subject will continue in the study. The Sponsor also has the 
right to discontinue the subject for protocol violations. The IRB may also have to be contacted 
if safety to the subject or if the scientific soundness of the study is involved. All protocol 
deviations must be documented in the CRFs. Protocol deviations will be tracked at the site 
level and (by the Sponsor) at the study level for the duration of the trial. 
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13 
ETHICS AND REGULATORY REQUIREMENTS 
This study is to be conducted in accordance with the requirements of this protocol and in 
accordance with principles consistent GCP, ICH E6, HIPAA regulations (45 CFR Part 164) 
and the Belmont Principles of respect for persons, beneficence, and justice. No protocol 
changes will be implemented without prior review and approval of the IRB, except where it 
may be necessary to eliminate an immediate hazard to a study subject. In such a case, the 
change will be reported to the IRB as soon as possible, according to IRB requirements. 
Additionally, all products used in this study are manufactured, handled, and stored in 
accordance with their FDA labeling. 
13.1 
IRB 
The Investigator at each center will provide the IRB with study materials, including but not 
limited to the clinical study protocol, ICF and any advertising materials. The study will not be 
initiated until the IRB provides written approval of the aforementioned documents and until 
approval documents have been obtained by the Principal Investigator and Sponsor or 
Sponsor designee. The site Investigator will not participate in the decision. If the Investigator 
is an IRB member, documentation must be provided indicating recusal from the approval 
process. Appropriate reports on the progress of this study by the Investigator will be made to 
the IRB as required by local and applicable government regulations and in agreement with 
policy established by the Sponsor. The Investigator is required to maintain an accurate and 
complete record of all written correspondence to and received from the IRB and must agree to 
share all such documents and reports with the Sponsor. 
No changes from the final approved protocol will be initiated without IRB prior written approval 
or favorable opinion of a written amendment, except when necessary to eliminate immediate 
hazards to subjects or when the change involves only logistics or administration. 
13.2 
Investigator’s Responsibilities 
The Investigators are responsible for performing the study in full accordance with the protocol 
and the current revision of the Declaration of Helsinki, the ICH GCP Consolidated Guideline 
and any applicable national and local laws and regulations. Information regarding any study 
centers participating in this study that cannot comply with these standards will be 
documented. 
13.3 
Subject Informed Consent Requirements 
Written and oral information about the study in a language understandable by the patient will 
be given to all patients by the Investigator and/or designee. Written informed consent will be 
obtained from each patient before any procedures or assessments that would not otherwise 
be required for the care of the patient are done and after the aims, methods, anticipated 
benefits, potential hazards, and insurance arrangements in force are explained, and the 
patient has been given sufficient time to ask questions and consider participation in the study. 
It will also be explained to the patients that they are free to refuse entry into the study and free 
to withdraw from the study at any time without prejudice to future treatment. It is permissible 
for a third person (e.g., a family member) to be present during the explanation of the study. 
The written ICF is to be in compliance with 21 CFR § 50.27 and GCP requirements. The 
Sponsor and/or designated CRO will approve the ICF and all amendments to the ICF prior to 
submission to the IRB. A copy of the ICF to be used will be submitted by the Investigator to 
the IRB for review and approval prior to the start of the study. Each study site must provide  
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the Sponsor with an unsigned copy of the IRB-approved ICF along with applicable 
documentation to support this approval. The original signed ICF is retained in the subject's 
study records and a copy are provided to the subject. A second copy may be filed in the 
subject’s medical record, if allowed by institutional policy. 
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14 
DATA HANDLING AND RECORD KEEPING 
14.1 
Recording and Collection of Data 
The primary source document for this study will be the subject's medical record. If separate 
research records are maintained by Investigators, the medical record and the research 
records will be considered the source documents for the purposes of auditing the study. 
Applicable source data will be manually transcribed to the approved CRF. The Investigator is 
ultimately responsible for the accuracy of the data transcribed on the forms. All source 
documents and CRFs will be completed as soon as possible after the subject's visit. 
The Investigator will review CRFs to indicate that, to his/her knowledge, they are complete 
and accurate. If further changes are made after this, the Investigator will need to again sign 
the Investigator signature page. Designated source documents will be signed and dated by 
the appropriate study personnel. The Investigator must agree to complete and maintain 
source documents and CRFs for each subject participating in the study. 
All research data will be entered, either electronically or manually, into a computerized 
database, designed in accordance with the clinical data manager. 
14.2 
Clinical Data Management and Monitoring 
The CRO will be responsible for the processing and quality control of the data. The study will 
be monitored by the CRO. Monitoring may consist of either on site or remote review of any 
and all applicable study records. All study CRFs and background source documents will be 
made available for monitoring. The study monitor will ensure that the investigation is 
conducted according to the protocol design and regulatory requirements. 
14.3 
Archiving 
All study documentation at the Investigator site will be archived in accordance with the study 
Clinical Trial Agreement and this protocol. 
Study records are subject to inspection by applicable health and regulatory agencies at any 
time. 
Records to be retained by the Investigator include, but are not restricted to: 
▪ 
Source data and the primary records upon which they are based (e.g., subject’s progress 
notes, AE data, test results and any other diagnostic procedures required to evaluate the 
progress of the study) 
▪ 
Signed protocols and protocol amendments 
▪ 
Laboratory results, ranges, and certifications 
▪ 
Product (SOC dressing supplies) and accountability records 
▪ 
Study personnel signature log 
▪ 
Correspondence to and from the Sponsor, designee, and IRB 
▪ 
Investigator and sub-Investigator CVs 
▪ 
Signed ICF and HIPAA consent forms 
▪ 
Subject screening and enrollment log 
▪ 
AE or SAE event reports 
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▪ 
IRB approval, re-approval letters and reports 
▪ 
Any documents pertaining to the conduct of the study 
These documents must be maintained and kept on file by the Investigator so that the conduct 
of the study can be fully documented and monitored. 
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15 
PUBLICATION PLAN 
The results of the study may be published in scientific literature and may also be used in 
submissions to regulatory authorities. It is the intent of the Sponsor and the Principal 
Investigator to publish or present the study results together with the other sites unless specific 
permission is obtained in advance from the Sponsor to publish separate results. Co- 
authorship with any of the Sponsor’s personnel will be discussed and mutually agreed upon 
submission of a manuscript for publication. 
All information concerning the Sponsor’s operations (such as patent applications, formulae, 
manufacturing processes, basic scientific data or formulation information supplied to the 
Investigator and not previously published) is considered confidential by the Sponsor and shall 
remain the sole property of the Sponsor. The Investigator agrees not to use it for other 
purposes without written consent. 
Publication and Disclosure: Because this is a multi-center study, sites and Investigators shall 
not independently publish, publicly disclose, present or discuss any results or information 
pertaining to site’s and Investigator’s activities conducted under this agreement, until such a 
multi-center publication is released under Sponsor’s direction; provided, however, that if a 
publication is not released within eighteen (18) months after completion of analysis of all study 
data from all sites within the multi-center study, site and Investigator shall have the right to 
publish the results of and information pertaining to site’s and Investigator’s activities 
conducted under this protocol and the clinical trial agreement with sponsor permission. Site 
and Investigator agree to submit any proposed manuscript, presentation, or other public 
disclosure regarding the study to Sponsor for review at least thirty (30) days prior to submitting 
such proposed manuscript to a publisher or delivering or making such presentation or other 
public disclosure to any third party. Within thirty (30) days of its receipt, Sponsor shall advise 
site and/or Investigator, as the case may be, in writing of any information contained therein 
that is confidential information (other than research results included in a proposed manuscript) 
or that may impair Sponsor’s ability to obtain patent protection. Sponsor shall have the right to 
require site and/or Investigator, as applicable, to remove specifically identified confidential 
information (but may not require removal of research results from a proposed manuscript.  
However, the Sponsor retains all the rights to use their own technical explanation to explain 
the research results which cannot be avoided by the Investigator) and/or to delay the 
proposed submission or delivery of the proposed manuscript or presentation or other public 
disclosure, for an additional sixty (60) days to enable Sponsor to seek patent protection. Site 
and Investigator shall not publish, publicly disclose, present, or discuss any results of or 
information pertaining to sites and Investigator’s activities prior to completion of the trial, even 
if the study is terminated before its completion and the final clinical study report is signed off 
or with respect to any endpoints or analyses, other than those specified in this protocol. 
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16 
REFERENCES 
1. 
Driver VR, Fabbi M, Lavery LA, et al. The costs of diabetic foot: the economic case for the 
limb salvage team. J Am Podiatr Med Assoc 2010;100:335-41. 
2. 
Rice JB, Desai U, Cummings AKG, et al. Burden of diabetic foot ulcers for Medicare and 
private insurers. Diabetes Care 2014;37:651-8. 
3. 
Margolis DJ, Kantor J, Berlin JA. Healing of diabetic neuropathic foot ulcers receiving 
standard treatment. A meta-analysis Diabetes Care 1999;22:692-54. 
4. 
Mauricio D, Piaggesi A, Ragnarson Tennvall G, et al. Predictors of lower-extremity amputation 
in patients with an infected diabetic foot ulcer. Diabetes Care 2015;38:852-7. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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17. SIGNATURES 
 
The protocol has been reviewed in respect to compliance with ISO 14155: 2020, applicable national 
regulations and with Encoll Corp’s SOPs. 
 
_____________________________________________________________________ 
Dr. Subra Guna, PhD 
 
 
 
 
 
 
 
Date 
Phone:  510-396-8581 
Email:  guna@encoll.com 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDICES 
Appendix A: Wagner Grades  
 
Grade 1: Superficial diabetic ulcer Grade 2: Ulcer extension 
• 
Involves ligament, tendon, joint capsule, or fascia 
• 
No abscess or osteomyelitis 
Grade 3: Deep ulcer with abscess or osteomyelitis 
Grade 4: Gangrene to portion of forefoot 
Grade 5: Extensive gangrene of foot 
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Appendix B: ABI or Arterial Doppler Ultrasound 
ABI Technique 
Place the subject in the supine position, with the arms and legs at the same level as the heart, 
for a minimum of 10 minutes before measurement, 
Select an appropriately sized blood pressure cuff for both the ankle and the arms (see figure 
below), the cuff width should be, at a minimum, 20% greater than the diameter of the 
extremity. The ankle cuff should be placed on the leg between the malleolus and the calf. 
Enough room should be left below both cuffs to permit placement of the ultrasound gel, so that 
the Doppler device can adequately detect the brachial, dorsalis pedis and posterior tibial 
arteries. 
Obtain the brachial systolic pressures of both arms. Use the higher of the arm pressures in the 
ABI calculation. Obtain the pressure in the dorsalis pedis and posterior tibial arteries for the 
extremity with the target ulcer. Use the highest pressure for the ABI calculation. 
Ankle-Brachial Index = Highest ankle pressure/ Highest brachial pressure. 
Care should be taken to cover the index ulcer during the ABI measurement. In addition, 
subjects should be informed that they may experience discomfort during the test, secondary to 
the pressure exerted by the cuff in the area of skin breakdown. 
 
Arterial Doppler Ultrasound Technique 
Place the subject in the supine position, with the arms and legs at the same level as the heart. 
Ultrasound gel will be applied at the ankle level to the anatomic position of the dorsalis pedis 
and posterior tibial vessels. The Doppler will then be applied to each vessel until a waveform 
is established, a printer will be used to document the Doppler waveform and at least five 
waveforms will be printed for each vessel. The clinician will verify biphasic or triphasic 
waveform. 
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Appendix C: SPP 
 
 
 
SPP can be employed to evaluate the subject’s vascular status. SPP is obtained using a laser 
Doppler. 
 
Technique 
1. Secure the laser Doppler flow sensor within the bladder of a blood pressure cuff equipped 
with a transparent polyvinyl chloride window for measuring microcirculatory perfusion during 
cuff inflation and deflation. 
2. Place the subject in supine position and keep still for five minutes. 
3. Apply the cuff to the proximal margin of the ulcer and inflate to 20 mmHg above the brachial 
systolic pressure. A stable laser Doppler output value near zero (< 0.1 volume %) should be 
reached before deflating. 
4. Deflate the cuff, first in 10 mmHg-stepwise decrements every five seconds to a pressure of 
50 mmHg, and then in 5-mmHg decrements every 15 seconds until the laser Doppler output 
increased for two consecutive pressure values. 
5. The pressure at which this first occurred is considered the SPP value. 
Subjects with SPP less than 30 mmHg have vascular insufficiency and are not candidates for 
enrollment. 
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Appendix D: TCOM 
Technique 
Place the subject in the supine position, with the arms and legs at the same level as the heart. 
Electrodes must be in contact with the tissue through the contact liquid. If there is air between 
the tissue and an electrode, TCOM values will be questionable. Erroneous readings may also 
occur if electrodes are placed directly over a bone or there is severe edema around the wound. 
For the best results, tests should be conducted at ambient temperature (21-23⁰) and the subject 
should not have smoked, nor had caffeine for several hours prior. 
 
 
1. Calibrate the TCOM electrode—this takes about 15-20 minutes. 
2. Clean the selected measuring site with alcohol or other skin-preparation solution. 
3. Dry the site well with a gauze pad. 
4. Take a standard fixation ring. 
5. Remove the fixation ring from the protective film. 
6. Apply the fixation ring to the measuring site as follows: 
• 
Press the center of the fixation ring onto the measuring site with a finger. 
• 
Run a finger around the rim circumference. 
• 
Press firmly to prevent leaks. 
7. Fill the hole in the fixation ring with 3-5 drops of the contact liquid. 
8. Affix the electrode into the fixation ring as follows: 
• 
Align the arrow on the electrode with one of the marks on the fixation ring. 
• 
Turn the electrode 90° clockwise to fasten it in the fixation ring. 
9. Repeat steps 1 to 8 if more electrodes are to be applied; note: several electrodes can be 
calibrated at the same time. 
It is sometimes advantageous to simultaneously use several electrodes placed strategically 
around the wound and calculate mean values from individual readings. 
The normal sequence of events for TCOM is measurement in air, the leg elevation test 
(optional) and the oxygen challenge. Shah et al20 determined that the optimal times for these 
events in terms of measurement time are 20, 5 and 10 minutes, respectively. 
 
 
 
 
 
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Appendix E: Debridement 
Many chronic wounds contain necrotic tissue that has a black or dark gray appearance. Wound 
eschar is usually full-thickness, dry devitalized tissue arising from prolonged ischemia and 
slough is an adherent fibrous material commonly creamy yellow in appearance. Chronic wounds 
can also have colonized bacteria in the form of biofilm or cells, particularly along the margins of 
a wound, which have stopped dividing - a process known as senescence. Finally, devitalized 
tissue and biofilms can harbor high levels of cytokines or cellular remnants that maintain the 
wound in the inflammatory or proliferative stages of healing, through cellular trafficking 
processes. Any of these factors can disrupt the normal stages of wound healing. 
Debridement is the process by which these elements are removed to permit healing, in effect 
changing the stalled chronic wound into an acute wound. In this clinical study, we have 
restricted the type of debridement to sharp debridement. 
 
Technique 
Sharp debridement comprises the following elements: 
1. The target ulcer and the surrounding skin are prepped with water or Saline. 
2. Anesthesia, topical or injected, is applied to the ulcer as necessary to reduce subject 
discomfort. 
3. Using a sterile technique, all non-viable tissue in the wound bed is excised using a scalpel 
and scissors. 
4. Excessive bleeding is controlled by using direct pressure, but cautery may be employed if 
necessary. 
If extensive surgical debridement is necessary during the run-in period (e.g., general anesthesia 
is required), the patient is not a candidate for this trial. Likewise, if other forms of debridement 
(e.g., enzymatic) are required during run-in, based on the opinion of the treating clinician, the 
subject should be screen failed. During the Treatment Phase of the trial, other forms of 
debridement are NOT permitted. If this should happen, the type of debridement should be noted 
in the CRF, using other forms of debridement besides sharp debridement more than once 
during the treatment phase will result in the subject being withdrawn from the trial. 
Although there is increasing evidence that more frequent debridement results in faster healing 
on average, the frequency and level of debridement in this trial is left up to the treating 
physician’s judgment. Excessive debridement can be as deleterious to healing as too little 
debridement. 
For wound bed preparation, follow the TIME principles: Tissue management (primarily 
debridement in this trial), control of Infection and Inflammation (management of infection, control 
of edema, management of exudate, etc.), Moisture imbalance (ensuring that the wound is at all 
times in a moist healing environment, as well as dealing with excessive wound exudate), and 
advancement of the epithelial Edge of the wound (addressing hypoxia, infection, desiccation, 
dressing trauma, hyperkeratosis and calluses, and cell senescence at the wound margin). 
(Dowsett C, Newton H. Wounds UK 2005; 1:58-70) 
 
 
 
8/6/2024 11:05:38 PM EDT
Subramanian Gunasekaran EnColl Corp