A Multicentric Randomized Controlled Clinical Trial Comparing the Use of High Purity Type-I Collagen-Based Skin Substitute vs. Dehydrated Human Amnion/Chorion Membrane in the Treatment of Diabetic Foot Ulcers
Phase: NA
Conditions: Diabetic Foot Ulcer (DFU)
Sponsor: Adichunchanagiri Institute of Medical Sciences, B G Nagara

Title: Multicentric RCT Comparing High Purity Type I Collagen-Skin 
Substitute vs dHACM in Treatment of Diabetic Foot Ulcers 
 
 
NCT No: NCT07046403 
 
Date: November 09, 2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

INTRODUCTION 
Diabetic foot ulcers (DFUs) represent one of the most serious complications of diabetes 
mellitus, affecting approximately 15-25% of diabetic patients during their lifetime [1]. It 
remains a major global health problem associated with infection, limb loss and increased 
mortality. The global burden of DFUs continues to escalate, with direct healthcare costs 
exceeding $30 billion annually in the United States alone [2]. In India, the prevalence of 
DFUs among diabetic patients is estimated at 6.2%, with regional variations ranging from 
5.6% to 9.5% [3]. The consequences of DFUs extend beyond immediate healthcare costs, as 
these wounds are associated with significant morbidity, prolonged hospitalizations, and 
increased mortality rates [4]. Recurrence is common and healing is often protracted despite 
best practice wound care. 
The pathophysiology of DFUs involves multiple interconnected factors including peripheral 
neuropathy, impaired circulation, biomechanical stress, and compromised immune function 
[5]. Traditional wound care approaches often prove insufficient, resulting in delayed healing, 
increased infection rates, and ultimately, amputation in 20% of cases [6]. The complex nature 
of diabetic wounds necessitates advanced therapeutic interventions that can address the 
underlying cellular and molecular deficits. 
Recent advances in regenerative medicine have introduced novel treatment modalities, 
including bioengineered skin substitutes and biological matrices, which have shown promise 
in accelerating wound healing and improving clinical outcomes [7]. Among these, high-
purity type-I collagen- based skin substitutes (HPTC) and dehydrated human amnion/chorion 
membrane (dHACM) have emerged as promising therapeutic options [8, 9]. Type-I collagen, 
comprising 97% structural similarity across species, provides an optimal scaffold for cellular 
attachment and tissue regeneration [10]. This bioengineered collagen matrix mimics the 
native extracellular architecture and creates a conducive microenvironment for cell migration, 
proliferation, and differentiation. The inherent non-immunogenic properties of highly 
purified type-I collagen, which are attributed to the absence of sulphur-containing amino 
acids that typically trigger immune responses, make it particularly suitable and well-tolerated 
for wound healing applications [11]. 
Conversely, dHACM utilizes the natural regenerative properties of placental membranes, 
which have been employed clinically for over a century [12]. The amnion and chorion 
membranes contain growth factors, cytokines, and extracellular matrix components that 
facilitate wound healing through anti-inflammatory and pro-regenerative mechanisms [13]. 

However, processing methods may affect the bioactivity of these products, potentially 
limiting their therapeutic efficacy [14]. 
Previous single-centre studies have suggested potential advantages of HPTC over dHACM in 
treating chronic wounds [15, 16]. However, the evidence base remains limited, with small 
sample sizes and single-centre designs limiting the generalizability of findings. Furthermore, 
most comparative studies lack comprehensive histological analysis to elucidate the 
underlying mechanisms of action. 
The objective of this multicentre randomized controlled trial was to compare the safety and 
efficacy of HPTC versus dHACM in treating DFUs, with particular emphasis on wound 
healing kinetics, histological parameters, and patient-centred outcomes. We desire to reiterate 
our previous single centre study result [17], that HPTC would demonstrate superior wound 
healing outcomes through enhanced cellular activity and improved tissue regeneration. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

MATERIALS AND METHODS 
Study Design and Setting 
This prospective, multicentre, randomized, controlled, two parallel group, open-label trial 
based on pooled design was conducted across four tertiary care centres with study protocol 
approved by the institutional ethics committees of all participating centres: Adichunchanagiri 
Institute of Medical Sciences (AIMS), B. G. Nagara, JSS Medical College and Hospital 
(JSS), Mysore and Mysore Medical College and Research Institute (MMC&RI), Mysore and 
Rajarajeswari Medical College and Hospital (RRMCH), Bangalore, all centres in the state of 
Karnataka, India. The study was done under the supervision of primary investigator Dr 
Naveen N (AIMS) with the other principal investigators included Dr Ravi Shivaiah, Dr Vijay 
Kumar and Dr Kamal Kumar M, respectively. The study protocol was approved by the 
institutional ethics committees of all participating centres and registered with 
ClinicalTrials.gov (Identifier: NCT07046403; Protocol Record Id: AIMS/IEC/206/2025). The 
investigators adhered to the applicable regulatory requirements and the trial was conducted in 
accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Written 
informed consent was obtained from all participants and patient confidentiality was 
rigorously 
maintained. 
 
Patient screening and eligibility 
The study population consisted of patients seeking treatment for DFUs. Eligible patients were 
those willing to participate and comply with scheduled visits on days 7, 14, 21, 28 
(intervention period) and 35 (follow-up period). The study included two phases: screening 
and treatment. The screening phase aimed to determine patient eligibility for the treatment 
phase. During the screening, a series of assessments were conducted, including 
demographics, medical history, concomitant medications, vital, signs, physical examination, 
foot ulcer history, clinical infection signs at the ulcer site, and ankle-brachial 
index measurement. 
Inclusion criteria: Patients aged 18-75 years with type 1 or type 2 diabetes mellitus presenting 
with chronic foot ulcers were screened for eligibility. Target ulcer size between 5.0-20.0 cm² 
measured post-debridement; ulcer duration of 4-20 weeks; adequate circulation documented 
by ankle-brachial index (ABI) 0.7-1.3; and glycated haemoglobin (HbA1c) <12%. 
Exclusion criteria included: Patients were excluded from the study if they met any of the 
following criteria: presence of active infection requiring systemic antibiotics, confirmed 

osteomyelitis or exposed bone in the wound bed, current use of immunosuppressive therapy, 
diagnosis of active malignancy, pregnancy or lactation, end-stage renal disease requiring 
dialysis, acute Charcot foot deformity, previous amputation that would affect target ulcer 
offloading capacity, and participation in other investigational studies within the preceding 30 
days. 
 
Randomization and Blinding 
Eligible patients were randomized 1:1 using computer-generated random sequence with 
variable block sizes (4, 6, 8) stratified by site. Allocation concealment was maintained using 
sequentially numbered opaque sealed envelopes. Due to the nature of the interventions, 
participants and investigators could not be blinded to treatment allocation. However, outcome 
assessors for wound parameters (photograph-based planimetry was performed by a central 
reader), histological analysis and quality of life measurements were blinded to treatment 
assignment. 
 
Interventions 
All participants received standardized wound care including debridement, infection control, 
glycaemic optimization, and pressure offloading using total contact casting or removable cast 
walkers. Subjects who met the study's inclusion criteria after the screening period were 
randomized into one of two groups: 
a. The HPTC arm (n=60) received high-purity type-I collagen-based skin substitute 
(Helicoll®, Encoll Corp, USA) that was carefully trimmed to precisely cover the 
wound bed, followed by application of standard of care (SOC) wound dressing. The 
reapplication schedule for HPTC was determined by the treating investigator's clinical 
judgment based on wound assessment and healing progress.  
b. The dHACM group (n=60) received dehydrated human amnion/chorion membrane 
applied according to manufacturer instructions with concurrent application of 
standard of care (SOC), with repeat applications performed as clinically indicated 
based on wound healing response. 
During the four-week treatment phase, patients were re-evaluated on days 7, 14, 21, and 28. 
The SOC bandage in both groups included an identical three-layer dressing systems: non-
adherent paraffin gauze (primary layer), absorbent gauze pads (secondary layer), and soft roll 
with crepe bandage (tertiary layer) - standardized 3-layer dressing system. 

If the study ulcer was found to be 100% re-epithelialized during the visit, no further study 
procedures were conducted at that time. The patient was then scheduled for a follow-up visit 
after one week to confirm the healing. If complete healing was not observed, an assessment 
was performed to check for signs of clinical infection. If an infection was diagnosed, 
treatment with topical antimicrobials (betadine, chlorhexidine) or oral antibiotics was 
allowed, but the use of topical antibiotics (erythromycin, polymyxin, mupirocin) was 
prohibited. 
Following the infection assessment, the ulcer was cleaned, photographed, and debrided at the 
investigator's discretion to ensure a clean, granulating ulcer base with minimal adherent 
slough. The SOC was then reapplied, and the patient was instructed to keep the bandaging 
dry. The patient was also advised to contact or visit the study site if the bandage became 
soiled or was removed. Topical antibiotics specifically on the wound were prohibited. 
Systemic antibiotics were permitted when clinically needed. 
 
Study completion 
Patients completed the study four weeks after their first treatment visit. However, if a 
patient's study ulcer closed before the four-week mark, they were considered to have 
completed the study at that time. Complete healing of the study ulcer was defined as 100% 
re-epithelialization with no drainage. Throughout the treatment period, patients had the right 
to refuse participation or withdraw from the study at any time without prejudice. If a patient 
chose to withdraw from the study, their last recorded wound measurement was carried 
forward and used to calculate the change in wound size and their final outcome. 
 
Outcome Measures 
Primary Outcome:  
1. Proportion of participants achieving wound size reduction at 4 weeks, measured using 
digital planimetry. Wound area was monitored at visits on days 7, 14, 21, 28 
(intervention period) and 35 (follow-up period) 
Secondary Outcomes: 
1. Histological assessment of vascular incursion and wound biology was performed 
using punch biopsies (2mm diameter) obtained from the wound edge on day 5 post-
treatment initiation. Histological parameters were evaluated using standardized 
scoring scales, including: 

a. vascular infiltration assessed on a 0-3 scale based on vessel count per high-power 
field,  
b. neo-epithelialization measured on a 0-3 scale based on epithelial migration 
distance from the wound edge,  
c. fibroblast activity quantified on a 0-3 scale based on α-SMA positive fibroblast 
count and cellular morphology,  
d. capillary density measured as vessels per mm² using CD31 immunohistochemical 
staining,  
e. inflammatory response graded on a 0-3 semi-quantitative scale, and  
f. collagen deposition assessed on a 0-3 scale using Masson's Trichrome staining.  
Additional secondary outcomes included  
2. Time to complete wound closure defined as 100% re-epithelialization with no 
drainage,  
3. Percentage wound area reduction measured weekly using standardized digital 
photography,  
4. Mean number of treatment applications representing the total applications required 
per participant during the 4-week treatment period,  
5. Adverse events including infection and other complications monitored throughout the 
study period,  
6. Quality of life assessed using patient-reported visual analogue scale (VAS) measuring 
change from baseline to follow-up week, and  
7. Scar quality evaluated using the Vancouver Scar Scale at 5 weeks post-treatment 
 
Vascularity assessment was done using biopsy on day zero of application to be compared 
with day five after the application of HPTC or dHCAM. For histopathological assessment, 
before application and on the fifth day after the application of either HPTC or dHCAM, a 2 
mm punch biopsy was obtained from the wound edge extending into the wound bed under 
local anaesthesia (2% lidocaine without epinephrine). Biopsy samples were immediately 
fixed in 10% neutral buffered formalin for 24 hours, processed through graded alcohol, and 
embedded in paraffin blocks. Serial sections of 4 μm thickness were prepared and stained 
with hematoxylin and eosin (H&E) for general morphology, Masson’s trichrome for collagen 
assessment, CD31 immunohistochemistry for capillary density evaluation, and α-SMA 
immunohistochemistry for fibroblast activity. Histological parameters that were evaluated 
included vascular infiltration, neo-epithelialization, fibroblast activity, capillary density, 

inflammatory response, and collagen deposition (Table 1). All histological assessments were 
performed by two independent pathologists blinded to treatment allocation. Inter-observer 
agreement was assessed using Cohen’s kappa coefficient. 
Table 1: Histological parameters evaluated in the ulcer bed at baseline and 
on day five of application 
Parameter 
Measurement tool 
Criteria 
Score 
Vascular 
infiltration 
Assessed by counting 
new blood vessels (0–3 
scale) 
Minimal vascular ingrowth 
(<5 vessels/HPF) 
0 
Mild infiltration (5–10 
vessels/HPF) 
1 
Moderate infiltration (11–20 
vessels/HPF) 
2 
Abundant infiltration (>20 
vessels/HPF) 
3 
Neo-
epithelialization 
Measured as epithelial 
migration distance from 
wound edge (0–3 scale) 
No epithelial migration 
0 
Minimal migration (<25% 
wound coverage) 
1 
Moderate migration (25–75% 
coverage) 
2 
Extensive migration (>75% 
coverage) 
3 
Fibroblast activity 
Quantified by counting α-
SMA positive fibroblasts 
per HPF and assessment 
of fibroblast morphology 
(0–3 scale) 
Sparse, inactive fibroblasts 
0 
Moderate cellularity, minimal 
matrix production 
1 
High cellularity, active-matrix 
synthesis 
2 
Very high activity with 
extensive matrix deposition 
3 
Capillary density Evaluated using CD31 staining, counted as vessels per mm² of tissue 
Inflammatory 
response 
Graded semi-
quantitatively (0–3 scale) 
Minimal inflammatory 
infiltrate 
0 
Mild chronic inflammation 
1 
Moderate mixed inflammation 
2 
Severe acute inflammation 
3 
Collagen 
deposition 
Assessed using Masson’s 
trichrome staining (0–3 
scale) 
Minimal collagen matrix 
0 
Loose, immature collagen 
1 
Moderate organized collagen 
2 
Dense, mature collagen 
architecture 
3 
*Data presented as scoring criteria; all parameters assessed using standardized histological 
grading scales 
 
 
 

Sample Size Calculation 
Based on previous studies, we anticipated 85% of HPTC patients and 55% of dHACM 
patients would achieve wound reduction. With 80% power to detect a 25% absolute 
difference in complete wound closure at 5 weeks, 5% significance level, and assuming 10% 
dropout rate, a total of n=120 with 60 participants per group were required, pooled in from 
across 4 centres. 
 
Statistical Analysis 
Statistical analyses were performed using SPSS version 28.0 (IBM Corp, Armonk, NY, USA) 
and R version 4.3.0. Continuous variables were expressed as mean ± standard deviation or 
median (interquartile range) based on normality testing using Shapiro-Wilk test. Categorical 
variables were presented as frequencies and percentages. 
Between-group comparisons for continuous variables used independent t-tests or Mann-
Whitney U tests. Chi-square or Fisher's exact tests were employed for categorical variables. 
Time-to-event analysis used Kaplan-Meier curves with log-rank tests. Mixed-effects models 
analysed repeated measures data. All analyses followed the intention-to-treat principle, with 
sensitivity analyses using per-protocol populations. A two-sided p-value of less than 0.05 was 
considered statistically significant for all analyses, with p-values less than 0.001 indicating 
highly significant differences between groups. 
 
Data collection and follow-up 
Demographic data, medical history, comorbidities, and baseline wound characteristics were 
recorded. Laboratory parameters, including haemoglobin, albumin, and glycated 
haemoglobin (HbA1c), were obtained at baseline and follow-up visits. 
Patients were evaluated at baseline, day 5, day 7, day 14, day 21, day 28, and day 35. At each 
visit, wound measurements were performed using standardized digital planimetry, with 
wound area calculated in cm². Histopathology examination results were recorded on baseline 
day 0 and day 5 after application. Digital photographs were taken for documentation and 
independent assessment. Complete wound closure was defined as 100% epithelialization 
without drainage or dressing requirements. Any adverse events, if they occurred, were duly 
noted and notified. Scar quality and durability were assessed at each visit, with patient 
satisfaction assessed at the fifth week, during the one-week follow-up. 
 
 

Ethical Considerations 
The study was conducted in accordance with ethical principles outlined in the Declaration of 
Helsinki. Written informed consent was obtained from all participants before enrolment. 
Participant confidentiality was maintained throughout the study period. Data safety 
monitoring was performed by an independent committee.