Pregnancy-specific biological substance adjuncts rejuvenating chronic non-healing ulcer

INTRODUCTION

The placenta, umbilical cord, and amniotic fluid with amniotic membrane are the pregnancy-specific biological substances most useful in regenerative medicine as a part of cellular therapy. Most often, pregnancy-related products are discarded after birth, but scientists have observed that the substances mentioned above have the potential to restore the function of damaged tissues. Screened pregnancy-related substance use may help for the reversal of wounds, such as non-healing ulcers against the background of burns and traumatic non-healing ulcers, pressure ulcers, diabetes, and leprosy. Scientists also hypothesized that the human umbilical cord is used for coronary artery grafting.^[1] The benefits of using these materials in regenerative medicine are immeasurable. Primarily, they are not complicated to collect by trained individuals. Second, these biological substances have various stem cells, cytokines, growth factors, and anti-inflammatory agents that support the healing process and maintain the normal function of an organ by repair and regeneration. Human umbilical cord blood was also tried to correct anemia.^[2] According to potency, totipotent cells may differ from all other types due to the capability of forming extra-embryonic tissue. Pluripotent stem cells, except chorionic and placental tissues, can create all cell types (endoderm, mesoderm, and ectoderm). Pluripotent is usually kindred with embryonic stem cells and is characterized by the expression of specific markers of octamer binding protein (OCT)-4 and stage-specific embryonic antigens (SSEA-4, SSEA-4 antigen [T-cell receptor alpha (TRA)]-4-60, TRA-1-81, Nanog, Rex-1-shy (SSEA-4 antigen [SSEA-4], SSEA-4 antigen [SSEA-4]-2).^[3,4] The production of induced pluripotent stem cells from differentiated human somatic cells was recently reported by retroviral transduction of significant transcription factors,^[5,6] which may help restore body functions. It has been observed that the placenta, a discretionary stem cell source, is supported by placental tissue derived from embryonic gastric cells, which have significant plasticity. Using pregnancy-specific agents contributes to the healing and regeneration of various wounds by liberating biological substances.^[7]

AMNIOTIC MEMBRANE

The amnion is a delicate yet durable structure that forms the underlying layer of the fetal membranes. It plays a vital role in protecting and supporting the developing fetus during pregnancy.

DEVELOPMENT OF THE MEMBRANE

The amniotic membrane develops early in pregnancy from the trophoblast layer of the blastocyst. By the 2^nd week of embryonic development, the amnion begins to form as a cavity between the epiblast and the trophoblast. This cavity eventually becomes the amniotic sac, which fills up with amniotic fluid. This sac cushions the fetus, regulates temperature, and allows for free movement, which is essential for musculoskeletal development.

STRUCTURE

It is made of three primary layers:

1. The innermost layer, which is a single layer of cuboidal epithelial cells

2. Basement membrane, a thin layer of epithelial cells, provides the structural support of epithelium

3. Mesenchymal layers, the outermost layer, comprise fibroblast-like cells and an extracellular matrix, giving the membrane strength and elasticity.

The amniotic membrane is vital for fetal development and has applications in regenerative medicine due to its anti-inflammatory, anti-scarring, and tissue-healing effects. Dr. John W. Davis was the first to report fetal membranes, specifically the amniotic membrane, as a surgical material for skin grafting. This pioneering work was published in 1910 when Davis explored its application in skin transplantation. His innovative approach laid the foundation for the broader use of amniotic membranes in various medical fields, including wound healing and regenerative medicine.^[8] The presence of bioactive molecules within the amniotic membrane, mainly cytokines (interleukin [IL]-10]), growth factors (transforming growth factor [TGF-β]), and tissue inhibitors of metalloproteinase (TIMPs), reduces inflammation and promotes wound healing.^[9,10] Several other uses of the amniotic membrane have since been reported surgically, including its use as a biological bandage for skin wound treatment, burn injury,^[11] and chronic leg ulcers.^[12] Amniotic membrane implantation was introduced in the field of ophthalmology in the 1940s to treat eye burns.^[13] Since then, the amniotic membrane has spread to reconstruct the ocular surface.^[14] In perforated wing bone surgery, glaucoma surgery, and keratopathy amniotic membrane used for restoration of normal function.^[15,16] The aim of preserving biological properties is to develop storage conditions that maintain the viability of these cells. Henner Bichler et al. have shown that cell life can be kept at 37°C for up to 28 days in room temperature pulp membranes or Dulbecco medium in L15 medium.^[17] Recently, attempts have been made to use sustainable amniotic cells in a scaffold-free tissue engineering approach using a novel cell blade strategy already in use with other cell types.^[18] However, the freshly collected amniotic membrane and fluid are slightly available and free, making it the best option for daycare centers.

HUMAN AMNIOTIC MEMBRANE-DERIVED CELLS

Recently, investigations suggested that it is a good source of various stem cells.^[19] Cells from the mesenchymal and epithelial zones of the amnion can be isolated easily by mechanical detachment of the amnion from the chorion, followed by digestion with trypsin or dispase to release the epithelial cells. In contrast, the mesenchymal cells can be let out through subsequent digestion with collagenase.

HUMAN AMNIOTIC EPITHELIAL CELLS (hAECS)

hAECs have been extensively investigated for their unique cell types in the amniotic membrane. Expression of embryonic stem cell markers such as SSEA-4, TRA I-60, and TRA-I-81^[20,21] as well as expression of molecular markers of pluripotent stem cells, including OCT-4, SOX-2, and Nanog, have all been reported for hAECs.^[22]

HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS (MSCs)

Different research groups have demonstrated that the phenotypic properties of hAMSCs are like those of bone marrow MSCs and have typical mesenchymal markers (CD73, CD90, and CD105). Still, hematopoietic markers (CD34 and CD45) and monocytes (CD14) are missing.^[23] Furthermore, hAMSCs do not express Human Leukocytic antigen (HLA)-DR and have low HLA-ABC levels, indicating potential applicability in clinical implant settings.^[24] Studies of the angiogenic potential of cells derived from amniotic membranes show basal expression of endothelial-specific markers fms-like tyrosine kinase 1 and kinase domain receptor (FLT-1 and KDR) and spontaneous differentiation of endothelial cells, further strengthened by VEGF exposure.^[25,26] Increased expression of these markers, alpha-FP production, and glycogen storage were observed after liver induction in ex vivo.^[25]

CELL STRUCTURE AND COMPONENTS OF THE AMNIOTIC MEMBRANE

The amniotic membrane is composed of three essential layers.^[23] The deepest translucent layer covers the embryo, or amniotic fluid layer, which covers the amniotic fluid. This amniotic layer is rich in mesenchymal stem cells, amniotic fluid epithelial cells, embryonic stem cells, and progenitor cells. The amniotic and chorion membranes have a basement membrane and current layer.^[27] The second or medium collagen-connected collagen tissue remains connected to the reticulocyte chorionic layer, which is rich in external collagen.^[28] The amniotic membrane is an abundant source of pioneering, fetal, pluripotent, and even pluripotent stem cells. The epithelial layer of the amniotic fluid membrane contains artificial epithelial Stem cells, which play a role in wound healing and recycling. Collagens IV, V, and VII and fibronectin, proteoglycans, glycosaminoglycans, laminin, and fibroblasts in the amniotic membrane provide strength and tension. This is a scaffold for cell migration in the wound area.^[29] The possibility of graft supply responses in amniotic fluid cell therapy is also low due to poor expression of HLA-A, B, C, and DR.^[30] Matrix metalloproteinases (MMPS), their inhibitors, and growth factors, help compensate for routine and unrestricted growth.^[31] Various growth factors, such as fibrous growth factor (FGF), platelet derived groth factor (PDGF), and TGF-β, promote healing methods that include anti-inflammatory molecules.

AMNIOTIC FLUID

It is a practical, nutritious liquid that surrounds the fetus during pregnancy. Amniotic fluid production begins mainly due to monochorionic membranes involving water’s simultaneous movement and active sodium and chloride transport through the fetal skin.^[32] Later, in the second half of pregnancy, most of the fluid results from urination and secretion through the fetus from the airway. Furthermore, the fetus swallows amniotic fluid and begins to secrete it through the intestinal tract of the stomach. Therefore, amniotic fluid composition is influenced by fluid dynamics and varies with gestational age.^[33] In addition to proteins, carbohydrates, fats, amino acids, enzymes, hormones, and pigments, fetal cells are also found in amniotic fluid. Amniotic fluid for prenatal diagnosis is routinely removed in a low-risk process (risk of miscarriage 1/200). Still, information on the origin and properties of cells in this fluid is limited. So far, various types of fetal cells have been found, including cells in all three germ layers.^[33] In addition to fully differentiated cells, progenitor cells and pluripotent embryonic stem cells were also detected. To better appreciate the origin and characteristics of these heterogeneous cell populations, several studies have been conducted using human amniotic fluid samples and animal models, focusing specifically on the regenerative ability of these cells and the possibility of using them to develop future cell-based therapies.

HUMAN AMNIOTIC FLUID CELLS

Amniotic fluid constitutes a very heterogeneous population containing fetal membranes and cells derived from the fetus. Amniotic fluid cells have long been categorized based on morphological, biochemical, and growth characteristics, with three major groups identified as colony image-forming amniotic fluid cells, epithelial-like, and fibroid cells.^[34] At the start of amniotic fluid-derived cell culture, both amniotic fluid and fluid-specific and epithelial-like cells were found. In contrast, fibroblast-like cells are cultured only by some, but not all, amniotic fluid samples. They can only be cultured by those that usually occur later in the cultural process. Although sheep liquid–liquid-specific cells are in cell culture, the epithelial-like cells are removed over time. Amniotic fluid-specific cells came from the fetal membrane, and epithelial-like cells were thought to be derived from the fetal skin and urine. In contrast, fibroblast-like cells of fibroblasts appear in fibrotic connective tissue and dermal fibroblasts.^[35] However, it has recently been suggested that amniotic fluid cells can be divided into two categories, adhering and non-adhering cells, using two-stage isolation and cultural protocols. Fibroblast-like cells are derived from mesenchymal cells and are non-adherent. This was confirmed by proteomics. This indicates the presence of proteins in differentiated cells such as epithelial cells, fibroblasts, keratinocytes, foreskin and epidermis, and mesenchymal cells.^[36] In addition to the presence of proteins commonly expressed in tissues such as the fetal liver, brain, heart, pancreas, cows, and eyes, the presence of proteins expressed in human embryonic stem cells was also observed in the sheep embryonic cells.

MULTIPOTENCY OF AMNIOTIC FLUID-DERIVED CELLS

It was described in the early 90s when a few cells were detected as hematopoietic cells,^[37] followed by the observation that amniotic fluid cells exhibited the properties of myogenic cells after culture with the rhabdomyoma cell line.^[37] Subsequently, they can successfully distinguish osteocytes, adipocytes, and fibroblast cells. Furthermore, some cells resembling amniotic fluid cells in BMS MSCs expressed CD90, CD105, CD73, and CD166. In addition, expression of CD49E, CD58, CD54, CD123, CD71, and CD44 was also detected, but hematopoietic markers such as CD45, CD34, and CD14 were not available in these cells. This phenotypic profile has been confirmed in other studies, indicating that amniotic fluid may be a new source of MSCs. One of the most essential properties of amniotic fluid during pregnancy is to cleanse the vaginal canal before the baby is born, destroying the pathogenic environment. Amniotic fluid is usually considered sterile and bactericidal. Few cells express pluripotent stem cell markers, such as SSEA-1, 3, 4, TRA-1-60, and TRA-1-81. Cells expressing these markers may correspond to embryonic and inducible pluripotent stem cells, but more characterization studies are needed. Studies have shown that these embryonic stem cell types have complex molecular behavior and can express all three germ layers. Like embryonic stem cells, these sheep cells also have high proliferation rates in vitro, with no teratoma or tumor formation reported yet. Another important stem cell component of sheep fluid is the stem cells of amniotic fluid, which can grow without a feeder layer. Under in vitro conditions, the self-renewal ability of these cells was observed to be relatively high. Like amniocytes, they do not form tumors.

MECHANISM OF WOUND HEALING THROUGH THE NEWLY COLLECTED AMNIOTIC MEMBRANE

Healing of the wound itself is a highly complex biological phenomenon. Three phases: (i) Generally accompany it. Inflammation, (ii) proliferation, and (iii) maturation by release of cytokines, growth factors, and stem cells. Amniotic membranes have several benefits and can be considered an excellent wound-healing model. The amniotic membrane can quickly stick to the wound bed and maintain a balance between neovascularization processes and control of mesenchymal stem cells, as well as the synthesis of MMPS and their inhibitors TIMPS, which promote a wide range of cell proliferation.^[38] This process facilitates the third stage. This helps re-express the wound surface for the epithelial cells of the amniotic fluid. Finally, amniotic membranes are assumed to inhibit proteases, PMN filtration, and growth factor secretion from donor fibroblasts. If previously used with various wounds, it is newly prepared for good results. Many researchers are suitable for clinical implantation. Bhattacharya et al. in 1989, the use of fresh AMS in patients with chronic venous ulcers failed by 11.1%. When using such membranes, there is always a risk associated with disease migration. Therefore, a proper donation test is required before the placenta is removed. Furthermore, the time interval between AM maintenance and implantation is very short. Many authors believe that the intact epithelial cell layer of cytokine pools and growth factors’ freshness provides better healing. Furthermore, the stem cell components of the membrane remain active and fresh, supporting correct healing.^[38] However, only a few studies have shown that lyophilized membrane grafts are more efficacious than fresh grafts. Cryopreserved amnion has also been proven effective, but there are also some failure cases. The frozen cumulus is also expensive. The stem cell components of membranes, cytokines, and growth factors are also intact and fresh. Simple availability makes it cheaper than all other preparations. Human AM stem cells have been shown to inhibit collagen-induced rheumatoid arthritis deposition by damaging joints with reduced NK cells and reduced arthritis index scores.

IMMUNOLOGICAL PROPERTIES OF AMNIOTIC MEMBRANE AND FLUID-DERIVED CELLS

The placenta forms the interface between the immunologically distinct mother and fetus. It has been suggested that cells from this tissue, including cells from the amniotic membrane, have immunomodulatory properties that maintain feto-maternal tolerance during pregnancy. Clinical studies support the hypothesis that fetal membranes are non-immunogenic, which can help treat skin wounds, burn injuries, and chronic leg ulcers; prevent tissue adhesion in surgical procedures; and reconstruct ocular surfaces. Moreover, several clinical trials in humans have proven that allogenic transplantation of amniotic membranes or hAECs without immunosuppressive treatment does not induce acute immune rejection. Studies investigating the immunological properties of human amniotic fluid-derived cells show that these cells weakly express HLA Class I and-G but not HLA-DR. Production of interleukin 6 (IL-6). This has also been observed during the culture of these cells, while expression of HLA-DR could be involved after treatment with interferon-γ.^[31]

CONCLUSION

Data acquired to date regarding cells derived from both the human amniotic membrane and amniotic fluid provide evidence in favor of their possible clinical applicability in the field of regenerative medicine in the future.

Many groups have reported that heterogeneous cell populations derived from the amniotic membrane or amniotic fluid can give rise to all three germ layers. This confirms that these cell populations at least contain progenitor cells and supports the possibility that they may also contain pluripotent stem cells. Applying newly collected, screened amniotic membranes and fluids was done because of potent progenitor cells and stem cells, combined with growth factors, and cytokines, unlike other methods for applying the amniotic membrane. Cases of a minor number of graft rejections post-amniotic membrane application, accompanied by a foul odor due to the sloughing and rejection of the amniotic membrane by the host’s immune system, and the presence of Pseudomonas aeruginosa have been reported. However, applying amniotic membranes and fluid to treat irreversible conditions has proved successful. Further, molecular studies are required to confirm the mechanism by which complex mechanisms such as regeneration, repair, and healing occur so effectively.

Last, we must consider the easy availability, cost-effectiveness, and superior biological dressing of newly collected amniotic membrane and fluid in chronic non-healing ulcers in this stem cell age.