European Journal of Cardiovascular Medicine

Burn wounds are the one of the most serious forms of injury, often leading to complex and systemic complications. Among them, the majority of burn cases are found to occur in low and middle-income countries like India with most of them hailing from rural areas.(1) Acute burn injuries followed by electrolyte imbalance, metabolic changes, infection, and circulatory shock are very challenging to manage and can lead to multiple organ failure. Treating burn wounds is challenging due to the risk of rapid microbial colonization, persistent inflammation, delayed epithelialization, and the formation of excessive scar tissue. In chronic or non-healing burns, inadequate blood supply, tissue necrosis, and repeated trauma to the wound bed further slow recovery. Effective management therefore requires not only protection from infection and fluid loss but also active promotion of tissue regeneration and functional restoration. Thus burn care and management especially critical care management and eventually proper rehabilitation adds up to a great amount of socio-economic burden. Also, the affected burn victim’s quality of life and mental and physical health gets miserably affected.

Sources of burn injuries –

Burn is an acute tissue traumatic injury causing loss of tissue integrity in the epidermal, dermal, or deeper underlying tissues due to close contact with (2) –

Thermal sources where the amount of injury depends on the temperature of the heat source, time period of exposure to the external thermal sources, and the skin’s thickness.

• Fire a source of dry heat can cause immediately a deep burn which can lead to a painful wound.
• Hot liquids or Scalds are sources of wet heat that initially appear to involve the epidermis and parts of the dermis but may also lead to partial-thickness and full-thickness burn injury. (3) Steam causes more severe burns than hot liquids as they contain more heat energy due to the latent heat of vaporization.
• Hot surfaces or hot metals are another common household source of dry heat which when in direct contact with skin can cause heat-induced burn leading to various levels of tissue destruction.

Chemical agents like acids, alkalis, or corrosive liquids burn or cause destruction of organic tissue and tissue integrity of skin by chemical action. The duration of exposure to those chemicals determines the extent of tissue damage. Burns caused by alkalis happen to be much worse than burns caused by acids. (4) Electrical agents like flash and lightning cause electroporation leading to deep tissue damage where the magnitude of tissue damage depends upon the strength of electric energy and the time period of electric current flow. (5) Radiation or radioactive energy can cause damage to the underlying tissue and due to their ability of DNA damage, they can sometimes lead to cancer formation. Sunburn, excessive radiation involving diagnostic procedures, and exposure to radiation working in the nuclear industry can cause deep skin burns. Friction causes traumatic injury to skin due to mechanical abrasion and a high amount of heat generated at the localized area due to sliding force by contact against some hard and rough surface. Generally, they generate superficial burns but sometimes can lead to second or third-degree burns causing deep tissue injury.

Classification of burn injuries –

Burn injuries cause local and systemic changes in tissues. In the case of first-degree burns, also known as superficial burns, the outer layer of skin or the epidermis is generally affected. Regarding second-degree burns known as partial thickness burns both the epidermis and part of the underlying layer of skin (or dermis) are involved. These types of burns are often accompanied by painful blisters. The affected region of the skin is usually red, moist with exudation and painful. The most serious case of burn the third-degree burn or full-thickness burn destroys both the epidermis and dermis damaging the underlying muscles, tendons, and even nerve endings.

Stages of burn wound healing –

The immediate step that occurs after skin suffers burn is the inflammatory stage. In this inflammatory stage, a cascade of mediators of inflammation and various growth factors play an important role in creating inflammation. Inflammation is a complex biological process in which there is facilitation of monocytes and neutrophil infiltration in the wound bed. (6-9)

The process of inflammation is followed by the proliferation stage and remodeling phase. (10) In the proliferation stage granulation tissue formation, re-epithelialization at the burn wound surface, and initiation of re-establishment of vascular network have been observed.

The final step of wound remodeling starts several weeks after the burn wound formation and the development of myofibroblasts from fibroblasts results in contraction of wound leading to scar formation. (11)

Objectives of the study

• One of the objective from clinical point of view of this work was to study the rate of promotion of healing by freshly collected amniotic membrane application as compared to normal conventional / saline dressing.
• In this study, one of the focus area was assessment of biomarkers and mediators related to wound healing to understand potentiality of screened and freshly collected hypo-antigenic amniotic membrane dressing in managing inflammation in non-diabetic chronic burn wounds as they probably have superior healing capacity owing to the presence of progenitor cells, cell based cytokines and growth factors.
• Biomarkers like IL beta 1 (IL-1β) and TGF beta 1 (TGF-β 1) are some of the key players for the scenario of inflammation, tissue remodelling and granulation tissue formation during stages of wound healing. Study on estimation and expression of markers and mediators involved with the inflammation during healing was one of main objectives in this study.
• Histopathological studies to understand the stages of wound healing of each burn wound.
• Whether freshly collected amniotic membrane can help in scar-less healing or healing with minimum scar formation was another interesting point on which importance was given to.
• Any adverse event like graft rejection in patients after the application of freshly collected amniotic membrane and whether there has been any secondary complications during dressing period had been monitored.
• Overall by continuing the study and maintaining follow-up of the patients, along with the abovementioned parameters, we are assessing whether freshly collected amniotic membrane can be a novel biological dressing model with cost effectiveness for the general mass having very limited financial capacity..

Initially 18 patients with varying degree of burn were randomly recruited for this present study. Out of them, 8 patients who disagreed with the protocol of application of freshly collected amniotic membrane at wound sites were transferred to the burn unit of a tertiary care hospital. The patients and one of their family members were counseled in their preferable language on the potentiality of pregnancy specific biological substances in treating burn and chronic wounds. After they agree with the treatment protocol they were asked for a voluntary written consent and their audio-visual consent in their preferable language were recorded as well. Also institutional ethical committee approval has been taken before the start of this study.

Inclusion and Exclusion criteria –

Before the start of the study certain criteria were followed for inclusion as well as exclusion of cases in this study. The criteria are summed up as follows -

• Both male and female patients of age group between 18 – 80 yrs were included in the study.
• Freshly collected amniotic membrane was applied on 10 patients (Sample group) and 10 patients were placed under Control group who received normal conventional burn wound dressing.
• All sero-negative patients tested for blood glucose, autoimmune disease, CBC (complete blood count), renal, hepatic, lipid and thyroid profile before inclusion.
• Excluded- Comatose, HIV (Human Immunodeficiency Virus) positive, Tuberculosis, any other immune compromised patients, patients with drinking, history of smoking or any substance abuse habits were excluded from this present study.

Now these 10 patients (8 male and 2 female) with 26%-65% of total body surface area burn calculated on the basis of famous rule of nine and as of then had developed wounds, were recruited in this study of application of freshly collected amniotic membrane (serologically screened for HIV-I & II, CMV, Syphilis, Hepatitis B & C, VDRL) at wound site. The affected area of the recruited individuals included both partial to full thickness burn. First of all amniotic membrane bathed in naturally sterile amniotic fluid were collected in sterile containers by caesarean section from sero-negative mothers with their consent. Placentas were not accepted from sero-negative mothers with the following conditions – a) premature rupture of amniotic sac; b) meconium stained pregnancy specific biological substances; and c) known history of pelvic inflammatory disease. Under sterile conditions the placental membrane was separated from placenta and if required amniotic membrane was separated from the chorionic membrane. These membranes are often rinsed with naturally sterile amniotic fluid to remove any blood clots or debris if any.

Before application of freshly collected amniotic membrane, wound debridement is done as per demand of a particular wound in patients. The burn wound sites of patients were then washed with sterile normal saline for removal of any dirt or debris followed by sprinkling of sufficient amount of clear amniotic fluid on burn wound sites. Within four hours of collection of placental membranes they were applied at the wound site of patients as freshly collected amniotic membrane was found to have rich support of cytokines and various growth factors. (12) The amniotic or the fetal side was normally used in case of partial thickness skin burn for early epithelialisation and maternal or chorionic side was applied for improving circulation via angiogenesis. Change of freshly collected amniotic membrane dressing is done in interval of every 7 days.  Such weekly dressing is carried out meticulously keeping in account of the size & depth of ulcer, reduction of signs of inflammation like redness, discharge, pain, local edema, induration etc. Quantitative pain assessement was done by Visual Analogue Scale in accordance with Thong,et al.,2018. (13)

Wound size measurement was done weekly based on –  wound area and percentage of wound contraction. Intially, exudation was seen from the wounds. The exudate was collected in sterile method and stored at – 20 C. Apart from them blood from      was collected after every 10 days from the patients. Both the stored exudates and the serum samples separated from blood were used for different biochemical and biomarker assessments. The two inflammatory biomarkers that play crucial role during wound healing are C-reactive protein (CRP) and Interleukin-6 (IL-6) were also assessed during different stages of wound healing. Wound fluids and serum samples were analyzed for some key biochemical parameters like Albumin, Lactate Dehydrogenase (LDH), Bicarbonate and total protein. Levels of IL-1β  and TGF-β1 were quantified by sandwich enzyme linked immunosorbent assay (ELISA). Precise monitoring of anti-inflammatory factors like TGF beta and pro-inflammatory factors like IL beta 1 helped to evaluate healing progression under the influence of freshly collected amniotic membrane dressing on chronic burn wounds. Samples from wound sites were taken by Punch Biopsy procedure and immediately transferred in formalin container. Histopatholgical studies were being conducting using these samples to understand the details of wound healing.

Dressing with freshly collected amniotic membrane shown decrease in wound size, rapid epithelialization and lowering of pain score. No mortality or adverse reaction had been noted. The wound region showed less discharge, lowering of pain, local edema and induration. Albumin level was found to be more in blood serum as compared to wound fluid and in both albumin level increased (from 36 gm/L to 43.5 gm/L in blood serum and 16 gm/L to 26 gm/L in wound fluid)  from non-healing to healing phase - Fig. 4(A). The LDH level that generally reflects the metabolic and injury status of wound tissue was found to be many times less in blood serum than that of wound fluid (from 389 U/L to 380 U/L in blood serum and 3820 U/L to 2650 U/L in wound fluid) - Fig. 4(B). Its decrease in level from non-healing to healing phase of wounds is also very much significant in wound fluids as compare to blood serum. Bicarbonate level increased slightly from non-healing to healing phase both in blood serum (29 mmol/L to 30.2 mmol/L) and wound fluid (16.4 mmol/L to 19 mmol/L) - Fig 4(C). This is relevant with maintaining the local wound environment slightly acidic. Total protein level increased from nonhealing to healing phase in blood serum (66.5 gm/L to 79.8 gm/L) and in wound fluid (30 gm/L to 47 gm/L) – Fig 4(D). Quantitative assessment of  anti-inflammmatory cytokines TGF beta 1 through ELISA indicated modulation of inflammatory response, increase in level of TGF beta 1 in mid-way of the experimental arm and return to its normal range near to the healing stage - Fig 1(b). Pro-inflammatory cytokine IL beta 1 level have been found to decrease around 3 months of amniotic membrane application. IL 6 and CRP level were quickly modulated by HAM and both of their level in blood serum increased initially but decreased drastically after 3 weeks of application.

• (b)

Fig. 1 – (a) Graphical representation showing better wound closure for the Amniotic membrane (AM) treated arm as compared to control arm.                              (b) Comparative representation of change in concentration level of TGF beta 1 with respect to progression of days in amniotic membrane (experimental) treated arm and control arm. It shows rise of TGF beta 1 concentration mid-way of AM application and back to normal range at around 4 months which may be linked with healing with minimum scarring but in Control group elevated level of TGF beta 1 around 4 months time may be linked with hypertrophic scar formation at wound site.

          (a)                                       (b)                                 (c)

• (b)                                    (c)

Some degree of hypopigmentation was seen in few cases which gradually reverted to normal skin color and architecture within 6 months follow-up.  Minimal scarring is noted as compared to Control group and contrary to our expectations not a single case of graft rejection had been encountered.

(A)

                                                                                                                                                                     (D)

Fig 4 : -  Representations on changes in the level of Albumin, LDH, Bicarbonate and Total protein  in both wound fluid and blood serum from non-healing to healing phases.

• (b)                                              (c)                                 (d)

Fig. 6 - Histological studies showing – (a)wound is in proliferative phase of healing  (b) Early to mid proliferative stage with presence of granulation tissue, presence of inflammatory cells, plasma cells, fibroblasts, new blood vessels, (c) Wound progressed into late proliferative phase, (d) wound is almost healed and in early remodeling phase.

The histological studies showed sequence wise healing of a chronic burn wound. In the field (a) - Granulation tissue occupied most of the field with persistent inflammatory infiltrate, mainly lymphocytes and macrophages with some neutrophils. Newly formed capillaries with thin endothelial lining were present. The epidermal layer was absent, indicating incomplete re-epithelialization. Plump fibroblasts and loose collagen were observed, showing active extracellular matrix deposition. No keratin layer was seen, suggesting the wound surface remained unhealed or partially healed.

In the field (b) –  chronic burn wound under treatment was progressing from the early-to-mid proliferative phase, where granulation tissue was actively forming. Fibroblasts were laying down the extracellular matrix, and angiogenesis was supplying nutrients for tissue regeneration. The wound had not yet progressed to the late proliferative or remodeling stages, where collagen becomes more organized and vascularity declines.

In the field (c) - There was active fibroblast proliferation with ongoing collagen synthesis. Prominent angiogenesis with a well-formed capillary network was observed. Persistent inflammation was present, and re-epithelialization was still in progress in some regions. The wound was not fully healed but was progressing towards closure, indicating it was in the late proliferative phase and transitioning to the remodeling stage.

In the field (d) - Minimal inflammatory infiltrate was seen with collagen deposition in the dermal area. A few small blood vessels with endothelial lining were present, suggesting neovascularization. Proper epithelialization had been established. All findings indicated that the wound was in the early remodeling (maturation) phase of healing and was very near to being healed (or healed)..

The use of amniotic membrane in cases of burn injuries has been documented since 1910 with the variable degree of success. (14) The advantages of amniotic membrane are mainly its water retention capacity, large in size to cover large wound areas, easy availability, requires simple preparation for wound dressing, can be easily sterilized and is hypo antigenic due to poor expression of HLA-A, B, C DR antigens on their surface. (15) Further Amniotic membrane have a cocktail of cytokines and growth factors like FGF, PDGF, EGF, TGF-Beta. (16) Amniotic membrane additionally contains matrix metalloproteinase and their inhibitors (TIMP’s) to counterbalance the

excessive growth. (17) Decrease in wound region pain had been observed in some patients of experimental group after 2^nd time dressing of freshly collected amniotic membrane and majority experienced less to minimum pain after 3^rd time dressing of freshly collected amniotic membrane.

This study demonstrates the sequential histological, biochemical, and cytokine changes accompanying chronic burn wound healing under treatment. Epithelial stem cells from the epithelial layer of the amniotic membrane help in re-epithelialisation and closure of large wounds. (16) Histology revealed a clear transition from an inflammation-rich, unhealed wound bed to a mature, remodeled tissue. The histological assessment of four sequential chronic burn wound specimens (as in Fig. 4) revealed a clear progression of healing from proliferation to maturation. In the earliest stage, the wound bed was dominated by granulation tissue with persistent inflammatory infiltrate, abundant plump fibroblasts, loose collagen, and active angiogenesis, with no epidermal covering, indicating incomplete healing. This was followed by an early-to-mid proliferative phase, where fibroblasts actively deposited extracellular matrix and newly formed capillaries supported tissue regeneration, though collagen organization and vascular regression had not yet begun. The third stage showed ongoing collagen synthesis, a well-developed capillary network, persistent inflammation, and partial re-epithelialization, marking the transition from late proliferation to remodeling. In the final stage, minimal inflammation, organized collagen deposition, few residual vessels, and complete epithelialization were evident, signifying early remodeling and near-complete or complete wound healing. This sequence reflects the expected histological evolution of chronic burn wounds under treatment.

Biochemical changes paralleled these histological findings. Serum albumin exceeded wound fluid levels but rose significantly in both compartments from non-healing to healing phases (serum: 36→43.5 g/L; wound fluid: 16→26 g/L), reflecting improved nutritional and wound status. Total protein increased similarly (serum: 66.5→79.8 g/L; wound fluid: 30→47 g/L). LDH, markedly higher in wound fluid than serum, decreased substantially during healing (3820→2650 U/L in wound fluid), indicating reduced local tissue injury. Slight bicarbonate increases in both serum and wound fluid suggested maintenance of a mildly acidic wound environment conducive to repair.

Cytokine profiling further supported the healing trajectory. Anti-inflammatory TGF-β1 rose transiently mid-phase, promoting fibroblast activity and angiogenesis, before normalizing near closure. Pro-inflammatory IL-1β declined markedly by three months post–amniotic membrane application, indicating resolution of chronic inflammation. Also by modulating CRP and IL- 6 level in blood serum, HAM helped in controlling inflammation leading to faster wound closure and reduced scarring. These patterns align with the known regenerative and immunomodulatory effects of amniotic membrane therapy.

Also amniotic fluid with which the amniotic membrane gets washed has anti-microbial properties and has a rich source of stem cells also known as amniotic fluid-derived stem cells which can grow without feeder layers, has a doubling time in about 36 hours, not tumorigenic in nature including greater self-renewal potency. (17), (18) So, this present work shows that proper use of this cost effective nature’s gift in the form of freshly collected amniotic membrane can help greatly in managing chronic burn wounds especially in a developing country like India with huge population pressure.

Human amniotic epithelial cells are generally considered to be hypoantigenic in nature and they have been shown to elicit beneficial effects on secretion of anti-inflammatory cytokines that suppress inflammation. This study highlights the value of combining histological evaluation with biochemical and cytokine profiling to monitor chronic burn wound healing. Sequential changes in tissue architecture—paired with measurable improvements in albumin, total protein, and reductions in wound fluid LDH—provided objective evidence of wound recovery. Cytokine modulation, particularly the mid-phase peak in TGF-β1 and delayed suppression of IL-1β, reflected the essential balance between inflammatory resolution and tissue regeneration. These findings strongly correlate with the histological phases of wound repair and underscore the therapeutic potential of amniotic membrane in accelerating healing. The multi-modal approach employed here offers a robust framework for both clinical monitoring and research into chronic wound management, reinforcing that biochemical and cytokine markers can serve as reliable adjuncts to histopathological assessment in determining wound healing status.

Also amniotic membrane washed in amniotic fluid has bacteriocidal property and hence rarely infection was observed at wound site. Amniotic membranes have been found to adhere firmly to wound surface.(19) Amniotic membrane dressing has proven to be a highly effective option for burn wound management. It offers multiple advantages, including significant pain reduction, shorter healing duration, and improved wound recovery outcomes.(20) In addition to its clinical benefits, it is easy to procure, cost-effective, and can be sterilized for safe use. So overall freshly collected amniotic membrane contains great potentiality to improve chronic non-healing wounds.

Acknowledgement – The support of  School of Tropical Medicine, Kolkata is duly acknowledged. The author declares no conflict of interest in this work.

Abbreviations –

HAM -  Human Amniotic Membrane

            TGF-β 1 – Transforming Growth factor- beta-1

             IL 6 – Interleukin 6

             CRP – C-reactive protein

             IL-1β  - Interleukin Beta 1

             CMV – Cytomegalovirus

            VDRL – Veneral Disease Research Laboratory

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