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Pregnancy-Specific Biological Substances (PSBS) appear during pregnancy to support the pregnancy process and is normally discarded to the trash/incinerator after the birth of the baby.The process of its aseptic collection is, however, easy and can be obtained on a regular basis by an obstetrician/trained physician or any other specific health personnel. After its proper screening for HIV (1& 2),Hepatitis(B&C), Cytomegalovirus(CMV), Syphilis, Malaria etc these Pregnancy Specific Biological Substances may be extremely effective if used as biological dressings in cases of cornea and pterygium degeneration, chronic burn injuries and diabetic foot ulcers within 4 hours of its collection.

Ontogeny of PSBS
Gross examination of the amniotic membrane reveals three important layers namely the amnion which is the innermost thin translucent layer covering the embryo followed by the lines of the amniotic cavity along with the amniotic fluid and a middle collagen-rich connective tissue forming layer which in turn is connected with an outer collagen-rich reticular chorionic layer. .The amnion consists of the mesenchymal stromal cells, amniotic epithelial cells including embryonal-like stem cells. Both the amnion and the chorion are composed of a stromal layer and a basement membrane.

Amniotic fluid is formed after 2 weeks of fertilization in the amniotic cavity and exists as a bag of water. It is composed of water, protein, lipid, fetal urine and electrolyte. Its main role is to help in the active transport of sodium and chloride, proteins and water through the chorio-amniotic membrane and the embryo’s skin. The volume of the fluid increases to around 270 to 400 ml between 16 to 20th week of pregnancy and about 800 ml in the 24th week. At times, the volume reduces to around 600 ml. This fluid may be used for karyotyping and genetic or molecular pre-natal diagnosis.

Amniotic Membrane
The use of amniotic membrane in cases of burn injuries has been documented since 1910. The advantages of amniotic membrane are mainly its water retention capacity, large in size to cover large wound areas and prevent the wound from exudation, easy availability, ease of dressing and sterilization, hypo antigenic due to poor expression of HLA-A, B, C DR antigens on their surface. However, it is not an absolutely immune privileged material for dressing as some degree of antigenicity and immunogenicity has been observed due to the presence of MHC Class I and II antigens. Amniotic membrane also has a structure quite similar to that of skin. Amniotic membrane has anti-microbial properties due to the presence of Beta-defensins and elafin, storage of lysozyme enzymes which can produce the bacteriostatic effect on gram-positive bacteria. It can also help in preventing the loss of proteins, water, electrolytes, and fluids by forming a biological dressing and cover the wound thereby giving a moist environment which is essential for healing.

Amniotic fluid
Amniotic fluid is another important component of the Pregnancy Specific Biological Substances (PSBS) that has the ability to prevent infection and help in regeneration through cell therapy. In a pregnant mother, amniotic fluid normally washes the vaginal canal before the birth of the baby so as to destroy any pathogenic environment. Therefore, this phenomenon explains the extreme sterile and bactericidal nature of the amniotic fluid. The viscosity of the amniotic fluid is an important property in topical application for burn injuries.

Cellular composition
Both the amniotic fluid and the amniotic membrane has a rich source of progenitor, multipotent, fetal and embryonal like stem cells. Epithelial stem cells from the epithelial layer of the amniotic membrane help in re-epithelialization and closure of large wounds. Apart from epithelial cells, fibroblasts are also present in the amniotic membranes providing lining and strengthening of the tissues thereby acting as biological scaffolds. Other important extracellular components of the amniotic membrane include fibronectin, proteoglycans, glycosaminoglycans, laminins, collagen type IV, V and VII which helps in maintaining the structural integrity of the tissue and acts as a biological substrate for migration of cells in advanced wound healing. Human amniotic epithelial cells or HAEC’s like amniotic membrane are also hypo antigenic due to the down-regulation of HLA-A,B,C,DR antigens with an increased suppression of neovascularization. Both amniotic fluid and the amniotic membrane have series of cytokines and growth factors like FGF, PDGF, EGF, TGF-Beta. Amniotic membrane additionally contains matrix metalloproteinases and their inhibitors (TIMP’s) to counterbalance the excessive growth. The amnion has also shown to contain mesenchymal stem cells that can transdifferentiate into cartilage, bone, heart and skeletal muscles, fat cells, neuronal cells and epithelial tissues following established in vitro cell culture protocols.

Amniocytes
The human amniotic fluid contains amniocytes which are a large pool of self-renewal cells, mainly fetal in nature with distinct pluripotent stem cell markers like SSEA-1, 3, 4 TRA-1-60, TRA 1-81. These amniocytes have an extremely complex molecular behaviour and express trophoblastic, ectodermal, endodermal and mesodermal cell, specific regulators. These are embryonic pluripotent like stem cells which are distinct from both embryonic and induced pluripotent stem cells have the propensity to be reprogrammed into a primitive fully functional pluripotent stem cell Amniocytes have a high proliferative capacity like human embryonic stem cells but do not form any tumours and are not immortal. Amniotic fluid 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. Lines maintained over 250 populations shows the AFSC’spluripotent nature which is due to the presence of long telomere ends in these AFSC’s found after normal karyotyping. Cost and affordability are another important major concern, especially in poor and developing nations. Freshly collected and properly screened amniotic membrane can be easily applied. In terms of hospital and patient economics, amniotic membrane & amniotic fluid due to its several unique properties can significantly reduce both patient infection control and cost to the hospital.

Mechanism
Wound healing is a complicated process accompanied by three important stages of inflammation, proliferation, and maturation with an initial release of different cytokines from the injured tissue. The four distinct and most important features of amniotic membrane are (a) Rapid adherence to the wound bed (b) Balance between angiogenesis by control of mesenchymal stem cells and TIMP’s (c) rapid re-epithelisation process due to the presence of amniotic epithelial cells and (d)Inhibition of protease enzyme and PMN infiltration and secretion of growth factors by the donor fibroblasts.

Ophthalmology
Roth and Sorsby in 1940 suggested the role of amniotic membrane application in ocular surgery in the management of second-degree chemical burns of the eye with extremely encouraging results. Tsubota and colleagues reported positive results with an amniotic membrane in 7 treated eyes with cicatricial pemphigoid and 2 of the four eyes with Stevens-Johnson syndrome. Further, Lee and Tseng reported the clinical use of amniotic membrane transplantation in 11 consecutive patients suffering from cornea ulceration between 14 to 17 weeks. Amniotic membrane grafts were sutured at the area of the defect and covered with a bandage. Ten patients healed without any recurrence for the next 6 to 9 months with the exception of one patient due to a presence of other background diseases. Kruse in 1999, conducted studies with the use of multiple layers of amniotic membrane for reconstruction of deep corneal ulcers where 9 out of 11 patients healed after 1-year follow-up. Hanada et al. used amniotic membrane as space fillers to cover the defect and let it act as a scaffold for the epithelium to grow. The study confirmed successful results in 8 patients with complete re-epithelialisation within 3 weeks time. Chen et.al used 3 layers of the amniotic membrane including an additional patch to treat neurotrophic ulcers secondary to diabetes, keratoplasty, radiation, HSV keratitis, and neuro-surgery. Twelve (12) patients showed improvement within 3 weeks. Similarly, Rakowska reported prompt healing in 7 patients with perforated corneal ulcers after application of amniotic membrane in 18 patients.

The first successful use of amniotic membrane in pterygium surgery was reported by Prabhasawat. Shimazaki used the amniotic membrane in 4 patients with primary and recurrent pterygium with satisfactory results. Further applications of the amniotic membrane along with steroid injection for primary and recurrent pterygia have shown encouraging results thereby promoting it as a useful alternative therapy to conjunctival autograft in pterygium surgery.

Amniotic membrane has also shown to exert the positive therapeutic effect on symptoms of bullous keratopathy by Pires and further follow-up of the clinical study further revealed post-operative pain-free post compared to intolerable preoperative application of amniotic membrane.

Application of PSBS in Burn
In burn injuries, death normally depends on the age of the patient, a percentage of the body surface burnt in the background of other diseases such as diabetes, hypothyroid, tuberculosis, nutrition level, and arthritis. Burn injuries in the background of other diseases like diabetes or Tuberculosis can also result in the formation of a chronic wound where high morbidity and mortality are observed due to wound sepsis, a disintegration of anatomic and functional integrity. After 3 months, if a wound has still not healed in a timely fashion with a functional integrity then it is termed as a chronic wound. Infection is also a major problem associated with burn-related injuries and can be controlled by dressing with artificial skin substitutes which should be ideally nontoxic, elastic with good adherence property and affordable. It should also facilitate the exchange of micronutrients, oxygen and prevent bacterial contamination. Unfortunately, none of the skin grafts fulfils the ideal parameters for an ideal requirement for burn injury. Between 1999-2009, 64 burn patients based on the famous rule of nine were randomly recruited for a study conducted by a research group at Calcutta School of Tropical Medicine. The process included treating all burn injuries with normal saline for removing the dirt and debris followed by the sprinkling of freshly collected and screened for Syphilis, CMV, Hepatitis B & C, VDRL, HIV 1 & 2 amniotic fluid at the site of the burn injury. This was followed by the application of the amniotic membrane as a biological dressing material. Stabilization of the healing procedures was followed by physiotherapy and rehabilitation. Routine follow-ups were conducted religiously in order to look for any offensive odour, exudation or any other clinical, local or systemic sign of infection. In long-term follow-up studies up to 6 months, no mortality was observed in any of the patients. Six patients reported problems of keloid and hypertrophic scars and the others reported complete healing who were further treated with amniotic membrane and appropriate release incision. Post leucoderma was not encountered in any of the cases. Hypopigmentation was seen in 14 cases but further follow-up studies showed normal skin colour and architecture.

Application of PSBS in Leprosy induced gangrene
The same group of researchers from Calcutta treated leprosy patients with gangrene by an amniotic membrane in 1999. All the cases were treated with removal of maggots, washed with normal saline for clearing the debris followed by the amniotic fluid as an antiseptic agent after 5 to 10 mins. The superficial or partial thickness wounds were dressed with freshly collected, full screened for HIV-1, 2, VDRL, Syphilis, Hep B,C & CMV amniotic membranes. Patients followed anti-standard leprosy treatment in the meantime. In all the cases healing was observed within 3 months of a follow-up study by a formation of granulation tissues and by re-epithelialisation followed by a further 6 months follow-up.

Application of PSBS Osteoarthritis
The osteoarthritis-related knee problem is a common morbidity observed among older patients. Freshly collected, properly screened amniotic fluid after necessary ethical committee clearance and patient informed consent was injected in 52 OA patients’ between 39 to 82 years of age who had not responded to any standard nonpharmacological and pharmacological treatment before. In this study, amniotic fluid was injected into the joint space with adequate antiseptic and aseptic preparations. It was a 2 arm study where one group received steroid treatment and the other amniotic fluid.The amniotic fluid treated group showed a better improvement when compared with steroid treated group after 2 months of treatment completion and this better therapeutic efficacy with amniotic fluid was observed till the 4th month.Also, while comparing joint effusion and non-joint effusion groups; it was revealed that in both cases where amniotic fluid was applied, a better positive outcome was observed when compared with intraarticular steroid injection.

Application of PSBS Paediatric burn
5 paediatric cases with varying degrees of burn (age range from 8 to 17 years) were also treated with freshly collected and screened amniotic fluid and fetal skin. (46) All these cases responded positively except two patients who had other conventional treatments from different hospitals.

PSBS & Cell therapy
Both amniotic fluid and the amniotic membrane have a rich content of different types of stem cells. In myocardial infarction rats, engraftment of amniotic fluid and membrane-derived stem cells has shown to differentiate into myocardial-like cells with increased venous ejection faction. In restoring liver function, AMSC and AF-MSC’s have shown the positive therapeutic effect after local and intravenous injection in animal models. Engrafted AM and AF derived mesenchymal stem cells have shown the increase in serum albumin level in rat models with severe lung disease. 8 weeks engraftment of AMSC’s has also been observed in liver tissue in a hepatic cirrhosis model. Decellularized AM has been successfully used as a nerve conduit for improving peripheral nerve regeneration in different animal models. AMSC’s have both reported to secrete different brain derived and neuro-derived trophic factors for regeneration of the central and peripheral nervous system. AM and AF stem cells can help in rescuing ovarian function in rats. The ovarian function improved after injection with AM and AF derived MSC’s through up-regulation of endogenous cytokines and creation of a perfect niche for follicular development. The level of E2 was found to be normal and upregulated with a normal downregulation of the FSH when compared to a normal mice ovary including follicular development was also observed to be normal.

Potentialities of PSBS
AFSC’s has the ability and has shown to enhance repair of damaged intestine in necrotising colitis via COX-2 dependent mechanism with improved clinical status, gut structure, and function.

The use of properly screened amniotic membrane and its fluid and its fresh application within 4 hours of its collection was first attempted in 1999.This process of freshly collected properly screened amniotic membrane has been utilized for a therapeutic process without any mortality since 1999. Reason behind the immediate use of the properly screened amniotic membrane is the utilization of its diverse biological properties. Both amniotic membrane and amniotic fluid are rich in growth factors, cytokines, and anti-inflammatory properties and it has been seen used within 4 hours of its collection after proper screening it can enhance and aid the rate of wound closure and healing.A very small number of graft rejection cases, accompanied with the foul odour, were observed which can be attributed due to the amniotic membrane sloughing and rejection of the host’s immune system or due to Pseudomonas aeruginosin.(58) Normally in such cases, the patients are often dressed with a new one.The use of amniotic fluid is essentially a new concept in the field of wound healing and is a rich hub of stem cell, cytokines and growth factors which play an important role in wound healing.