Scientific Papers

A single-center, open-labeled, randomized, 6-month, parallel-group study to assess the safety and efficacy of allogeneic cultured keratinocyte sheet transplantation for deep second-degree burn wounds: rationale and design of phase I/II clinical trial | Trials

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Study setting {9}

This study will be conducted at Shahid Motahari Hospital, Iran University of Medical Sciences, Tehran, Iran. Shahid Motahari Hospital is one of the burn referral hospitals in Iran. Therefore, the composition of the population referring to this hospital shows the composition of burn patients in the whole country.

Eligibility criteria {10}

Prior to the commencement of any study procedures, patients are required to sign the written informed consent.

The following are the inclusion criteria:

  1. 1)

    Age between 18 and 70 years

  2. 2)

    Both genders

  3. 3)

    Acute second-degree burn injuries involving 20–50% of the total body surface area (TBSA)

  4. 4)

    At least one burn injury for phase 1 and three burn injuries for phase 2 with a maximum area of 60 cm2 in the trunk or limbs, with intact musculoskeletal tissue

The following are the exclusion criteria:

  1. 1)

    Having underlying diseases which affect wound healing such as cardiovascular disease, diabetes, malnutrition, and hypertension

  2. 2)

    Pregnancy or breastfeeding

  3. 3)

    Active infection, malignancy, or immunosuppression

  4. 4)

    Respiratory tract injury

  5. 5)

    Having additional trauma injury such as fracture or CNS trauma

  6. 6)

    Need for intensive care

  7. 7)

    Unwillingness to cooperate in the intervention and follow-ups

Who will take informed consent? {26a}

All participants will sign informed consents after an accurate explanation of the process by the physician (S.F.)

Additional consent provisions for collection and use of participant data and biological specimens {26b}

N/a. All necessary items have been mentioned in the main informed consent, and there is not any ancillary study.

Interventions

Explanation for the choice of comparators {6b}

Autologous skin transplantation or conventional treatment with Vaseline gauze and antibiotics have been considered as comparators due to the fact that these therapeutic methods are the most common standard treatments.

Intervention description {11a}

In the first phase of the study, following initial resuscitation, patients will receive allogenic transplantation within the first 5–10 days after the injury. After preparing the patient for the surgery and through general anesthesia in the operating room of Shahid Motahari Hospital, Tehran, Iran, debridement of the necrotic tissues will be conducted by the tangential method. After that, an adrenaline-soaked gauze will be placed on the wound site for 3–5 min to control the bleeding. After preparing the underlying condition, a 2nd-degree burn wound with a maximum area of 60 cm2 will be selected and will be covered by an allogeneic keratinocyte sheet. In the second phase of this project, the same procedure will be conducted; however, based on the randomization list, three separate wounds of a single patient will be selected, and allogeneic keratinocyte sheet transplantation, autologous skin transplantation, or conventional treatment will be selected for each wound. Encountering any serious adverse events (SAEs), the second transplantation of the keratinocyte sheet will not be administrated and the patient who experienced SAEs will receive conventional treatment. Figure 1 provides a flow diagram for phases I and II of this protocol. In addition, the SPIRIT flowcharts of the study are presented in Figs. 2 and 3.

Fig. 1
figure 1

Patients flow diagram phase I and II

Fig. 2
figure 2

SPIRIT flowchart of phase I

Fig. 3
figure 3

SPIRIT flowchart of phase II

Criteria for discontinuing or modifying allocated interventions {11b}

Once any reports of SAEs related to the product transplantation are documented, this trial will be terminated.

Strategies to improve adherence to interventions {11c}

The patients will be monitored by regular follow-ups on days 3, 7, 10, 14, 21, and 28, after the transplantation. In addition, in the 3rd and 6th month after the transplantation, we will also have additional follow-up sessions which could provide an excellent adherence to the intervention.

Relevant concomitant care permitted or prohibited during the trial {11d}

During the study, the patients are allowed to use medications such as non-steroidal anti-inflammatory drugs (NSAIDs) for symptom alleviation.

Provisions for post-trial care {30}

N/a. There is no ancillary study and post-trial care. The patients in phases 1 and 2 will be followed up for 6 months after the first transplantation.

Outcomes {12}

The primary endpoint for this study is safety, which will be evaluated in all follow-ups by Common Terminology Criteria for Adverse Event (CTCAE), version 5.0 [27]. Furthermore, secondary endpoints of this project are time to wound epithelialization, skin graft take rate, scar assessment, wound closure, and quality of skin structure.

Participant timeline {13}

The study timeline for phases I and II are presented in Figs. 4 and 5, respectively.

Fig. 4
figure 4

Follow-up timeline of the phase I study

Fig. 5
figure 5

Follow-up timeline of the phase II study

Sample size {14}

To determine the necessary sample size for the study comparing the response rate between the control group and the intervention group, the formula for duplicate sizes in the two groups was used. It was assumed that four rounds of transplantation would be performed, with a mean correlation of 0.5 between repetitive interpersonal measures, a 5% first type of error, a statistical power of 95%, and a minimum difference in response rate of 45% based on similar studies [26,27,28,29]. Using these parameters, a sample size of 40 patients was estimated. In the first phase of the study, 5 patients will be included and in the second phase, and 35 patients will be enrolled based on the approved inclusion and exclusion criteria.

$$n=\frac{{2\left( z_{\alpha/2}+ z_{\beta }\right)}^{2}{\sigma }^{2}\left\{1+\left(m-1\right)\rho \right\}}{{md}^{2}}$$

Recruitment {15}

Given that Shahid Motahari Hospital serves as a prominent referral center for burn cases in Iran, the considerable volume of patients meeting our specific inclusion criteria is noteworthy. As a result, the process of patient recruitment is anticipated to transpire within the projected timeframe.

Assignment of interventions: allocation

Sequence generation {16a}

In phase I of the study, we will enroll eligible patients who meet the inclusion and exclusion criteria. This will be done through an open-label approach. In phase 2, random allocation software will be used as the method for randomization. By means of random allocation, each patient’s wounds will be assigned to one of the three groups: group A (autograft), group B (conventional treatment), or group C (keratinocyte sheet). This allocation will follow a 1:1:1 ratio and will adhere to a computer-generated randomization schedule that considers specific strata based on the wound site. Various wounds within a single patient will be subjected to this analysis. The investigation will involve a comparative analysis of the safety and efficacy of allogeneic skin grafts in contrast to autograft skin transplantation and conventional treatments, which encompass the use of Vaseline dressings and topical antibiotics.

Concealment mechanism {16b}

The mechanism of implementing the allocation sequence is central randomization. In this study, allocation of participants is performed centrally by a designated system (which generates a randomized list for allocation of patients in different groups) not directly involved in the recruitment or assessment of participants. Central randomization was implemented to ensure unbiased allocation of participants to treatment groups.

Implementation {16c}

The sequence of allocation will be conducted by an investigator (Dr. H.M.). Patients’ enrollment will be done by Dr. M.D. Assignment of patients to the groups will be done by Dr. S.F.

Assignment of interventions: blinding

Who will be blinded {17a}

N/a: this study is open-label so there is no blinding.

Procedure for unblinding if needed {17b}

N/a: this study is open-label so there is no blinding.

Data collection and management

Plans for assessment and collection of outcomes {18a}

Patients will be followed up on days 3, 7, 10, 14, 21, and 28, after the transplantation. In addition, in the 3rd and 6th months after the transplantation, we will also have additional follow-up sessions. In each follow-up session, a photo of the patient’s wound will be taken. The degree of epithelialization will be evaluated in each follow-up session through photography by the ImageJ software and at least three repetitive images for each wound will be taken. The skin graft take rate will be assessed on the 10th-day visit, and in the context of < 50% graft take, the allogenic transplantation will be repeated. In the 3rd and 6th month follow-ups, the scar and wound closure assessment will be conducted based on the Vancouver Scar Scale (VSS) and the Patient and Observer Scar Assessment Scale (POSAS).

Plans to promote participant retention and complete follow-up {18b}

Retention strategies

The study will implement a comprehensive retention strategy encompassing regular and frequent follow-up sessions. These follow-ups will involve diverse laboratory tests and complimentary visits. In addition, participants will receive written feedback regarding the outcomes of the conducted health screenings.

Withdrawal protocols

Participants hold the prerogative to withdraw from the study at their discretion, irrespective of the rationale, and at any juncture. Furthermore, the principal investigator retains the authority to withdraw participants from the study if it is deemed necessary for their safety or if they are unwilling or unable to adhere to the stipulated study protocols. The possibility of participant withdrawal also exists in the event that the study is prematurely terminated by the study sponsor, governmental bodies, or regulatory authorities prior to the planned culmination date. It should be noted that the discontinuation of the study product for any reason does not constitute grounds for withdrawal from the study.

Data management {19}

The data will be collected based on a research-made case report form (CRF). The CRF will include study timeline, history of present illness, basic laboratory data, CTCAE form, photographic and graft take rate information, standard questionnaires of VSS and POSAS, and also a form for pathological evaluation for phase one of the study.

Confidentiality {27}

All personal information of the patients will be collected and maintained by a dedicated investigator at Royan Institute in order to protect confidentiality.

Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}

In the phase 1 clinical study, 6 months after transplantation, a skin biopsy with a 2-mm2 diameter will be taken from three areas: areas treated with keratinocyte sheets, areas treated with autograft, and surrounding healthy skin. The samples will be dehydrated by formalin and ethanol, and after being placed in paraffin wax, they will be cut using a microtome with a thickness of 5 µm. Our samples will be stained with hematoxylin and eosin (H & E). The skin texture, skin ultrastructure, including hemidesmosomes, different tissue layers, and cellular infiltration will be assessed through microscopic evaluation. The collagen composition will be visualized by Masson’s Trichrome (MT) staining. In order to check the epithelialization, vascularization, fibrosis, and scarring, the immunohistochemical staining of tumor growth factor-beta (TGF-β) 1 and 3 will be performed. At least three images will be taken from each sample under the microscope. Due to the pathologic report and stored images, the samples will not be stored.

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