Amount and distribution of selected biologically active factors in amniotic membrane depends on the part of amnion and mode of childbirth. Can we predict properties of amnion dressing? A proof-of-concept study

Introduction

The amniotic membrane (AM), the innermost layer of fetal membranes, that protects the embryo during pregnancy, is a valuable biological material with a wide range of applications in clinical medicine. In tissue engineering AM serves as a scaffold supporting cell growth and migration [1, 2]. It has been used as a graft for ocular surface reconstruction, pleural and pericardial closure and as a conduit for peripheral nerve regeneration [3-7]. Several studies have shown the clinical usefulness of amnion in wound management, especially in treatment of severe burns and chronic ulcers [8]. However, results of these studies, especially in regards to treatment effectiveness, differ significantly between reports and between treated individuals [9, 10]. Several studies have shown that fresh, as well as sterilized amnion specimens, contain a wide range of various cytokines, including growth and differentiation factors [11, 12]. It is plausible that observed alternate influence of amniotic membrane may be due to the variable content of mentioned factors between amnion samples originating from different donors. Indeed, in our previous pilot study we have assessed amniotic membrane samples originating from different donors using the proteomic microarray system [13]. Results of this study have revealed certain differences in profile of biologically active factors among samples obtained from different donations. Moreover, some authors reported variations concerning the AM morphology and histological structure depending on the specific region of each AM. Usually, three different areas of the amnion: cervical (apical), placental, and a mid-zone, are distinguished [14]. The cervical area is a region of the membrane overlying the cervix, where the amnion ruptures prior to delivery. Significantly thinner and weaker than the rest of the membrane, with less epithelial cells, it is often described as a “zone of extreme morphology” [15-18]. In addition to that, some variations in the content of several biologically active factors involved in tissue regeneration, including epidermal growth factor (EGF), transforming growth factor  (TGF-) and matrix metalloproteinase (MMP)-9, between cervical and placental area of the AM have been reported so far [19-21]. Furthermore, recent studies suggest that biological properties of the amniotic membrane may also depend on duration of gestation, as well as the mode of childbirth [22, 23].
Therefore the aim of the present proof-of-concept, screening study was to assess and compare the content of selected biologically active factors involved in tissue regeneration in the amnion samples originating from cervical and placental area. In order to clarify, whether the content of these factors may depend on the method of delivery, samples obtained from elective cesarean sections, as well as on-term natural (physiological) deliveries have been assessed.

Material and methods

Amniotic membrane samples collection and preparation

Amnion samples have been collected in the Obstetrics and Gynecology Department of Warsaw Medical University Hospital. A total number of 7 membranes have been collected: 4 AM originating from spontaneous deliveries at term, 3 AM originating from elective cesarean sections. Written informed consent was obtained from all donors of amnion samples. The concept of the study was reviewed and approved by the local bioethics committee. The study procedures conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki.
From each amniotic membrane sample a cervical and placental portion has been separated, according to the method described by Moore et al. [21]. Shortly, the placenta and its membranes, collected after the delivery, have been placed on a cutting board and cut, perpendicularly to the placenta surface, up to the placental rim. Once the fetal membranes lay flat, samples have been taken from the area overlying the placenta surface and from the region most distant from placenta, identified as a cervical area of the membrane. Then, the amnion membranes have been separated from decidua layer, and cut into fragments of approximately 1 cm2, that have been used to prepare amniotic membrane extracts.
Subsequently amniotic membrane samples have been immersed in 1% Triton X-100 in phosphate buffered saline, supplemented with cOmplete Mini, a mixture of broad range protease inhibitors (Roche Diagnostics, Mannheim, Germany), and mechanically dispersed using glass homogenizer. The obtained suspensions have been centrifuged, and supernatants were collected into fresh tubes, aliquoted and stored until being used for further analysis.

Protein microarray

The assay was performed using the Proteome Profiler kit (R&D Systems Inc. Minneapolis, USA) as described in manufacturer’s protocol. The 200 µl of standardized amniotic membrane extracts were mixed with a cocktail of biotinylated detection antibodies and applied onto nitrocellulose membranes spotted with respective capture antibodies. After the overnight incubation, at 8°C, the membranes were washed and then incubated with streptavidin-horseradish peroxidase conjugate, followed by use of the chemiluminescence detection system. Finally, the membranes were exposed to the X-ray film (Agfa-Geavert, Mortsel, Belgium) for 5-15 min, to achieve an optimal intensity of the signal. After development the film was scanned, and the optical density of each analyzed spot was assessed using GelWorks 2D software (UVP, Cambridge, UK). Then, optical density of respective factors on each membrane was compared to optical density of positive control spots and their relative amount in amnion samples was expressed as a sample-to-control ratio.

Results

In all of the amnion samples high amounts of angiogenin (ANG), insulin-like growth factor-binding proteins (IGFBP)-1, -2 and -3, as well as serine protease inhibitor (Serpin) E1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) have been detected. The mean and median concentrations of these factors in amniotic membrane samples derived from cesarean sections and physiological deliveries are presented in Table 1.
All amniotic membranes derived from elective cesarean sections were characterized by lower mean amount of ANG, IGFBP-2, IGFBP-3 and TIMP-1 than membranes obtained after physiological deliveries. The mean levels of IGFBP-1 and Serpin E1 were similar in both membrane groups.
The intra-donor variations in distribution of selected biologically active factors between cervical and placental region of each membrane were also observed, as shown in Fig. 1. In the group of membranes derived from elective cesarean sections the mean amount of ANG, IGFBP-1, IGFBP-2, and Serpin E1 in the placental region was higher than in the cervical portion of AM. The levels of TIMP-1 in placental region were slightly lower than in cervical parts of these membranes. In samples from physiological deliveries higher amount of ANG, IGFBP-1, -2, and -3, as well as TIMP-1, was observed in placental region, whereas the level of Serpin E1 was higher in cervical region of these membranes.
Placental portions of all amniotic membranes derived from elective cesarean sections were characterized by lower mean amount of ANG, IGFBP-2, IGFBP-3 and TIMP-1 than corresponding region of membranes obtained after physiological deliveries. The mean levels of IGFBP-1 and Serpin E1 were similar in placental region of both membrane groups. Samples obtained from cervical area of membranes from elective cesarean section contained less ANG than corresponding samples of membranes from physiological delivery, whereas the respective mean levels of IGFBP-1,-2,-3, Serpin E1 and TIMP-1 in cervical region were similar in both groups.

Discussion

The results of this screening study have shown that the amniotic membranes obtained from physiological delivery contained larger amounts of selected pro-angiogenic and growth factors, i.e. ANG and IGFBPs than amniotic membranes from cesarean section. Noteworthy, mentioned biologically active factors were present predominantly in the placental region of amniotic membranes from both groups. All mentioned biologically active factors, found in assessed samples of amniotic membrane, may contribute to its beneficial actions as a dressing material. These factors may stimulate cell growth, proliferation and migration of fibroblasts, epithelial cells and vascular endothelial cells into the wound bed, and thus may actively promote wound healing [24].
Insulin-like growth factor-binding proteins are family of factors, which regulate half-life, availability and activity of Insulin-like growth factors (IGFs). It has been proven that co-administration of topical IGF-1 and IGFBP-1 or IGFBP-3 at wound sites enhanced stimulating effects of IGF-1 on re-epithelialization and granulation tissue deposition. The IGFBPs display also some IGF-independent activities like regulation of cellular migration, proliferation, and pro-apoptotic activity. These properties of IGFBPs can be connected with interaction with TGF- 3 receptor [25]. In our study IGFBP-1, -2, -3 were present in all amnion samples, and their amounts in membranes collected from natural deliveries were higher as compared to those from cesarean section. In both groups quantities of IGFBPs were higher in placental region than in cervical portion of amniotic membrane.
ANG, a plasma protein belonging to the ribonuclease A superfamily, is considered to be one of the most potent inducers of angiogenesis in vivo. By binding to smooth muscle and endothelial cells it activates the production of several proteases and plasmin that degrade the basement membrane components. This allows the endothelial cells to penetrate and migrate into the perivascular tissue [26]. In our study, similarly to IGFBPs, ANG in placental region was higher than in cervical portion of amniotic membranes, especially in samples from natural delivery.
The family of TIMPs consists of four protease inhibitors, with different affinity to various MMPs, e.g. TIMP-1 preferably binds to Membrane Type-MMPs, whereas other TIMPs are less selective. TIMPs are secreted by different types of cells, like macrophages, vascular smooth muscle cells and platelets. They suppress the MMPs activity by binding to their catalytic domain and blocking enzymatic activity [27]. Since an excessive expression and hyperactivation of MMPs, particularly MMP-2 and MMP-9, has been proven to be one of pathomechanisms of delayed wound healing, the presence of MMP inhibitors in a wound environment is important for effective wound treatment [28,29]. In our study the presence of TIMP-1 was confirmed in all assessed amniotic membrane samples. Median amount of TIMP-1 was higher in physiological delivery group than in cesarean section group (2.17 vs. 1.39 respectively). Comparable median amounts of TIMP-1 between cervical and placental area of the membrane were obtained in both: physiological delivery and cesarean section groups.
Serpin E1, also known as a type-1 plasminogen activator inhibitor (PAI-1), primarily produced by endothelial cells, is the member of serine protease inhibitor gene family. It acts as a main inhibitor of two serine proteases, tissue plasminogen activator (tPA) and urokinase (uPA), inhibiting fibrinolysis [30]. It has been proven that serpin E1 facilitates migration of in vitro cultured cells [31]. In vivo studies on animal model have demonstrated that the expression of serpin E1 increased rapidly at the site of injury and remained elevated up to the formation of keratinocytes monolayer on the damaged skin surface [32]. Sustained high level of serpin E1 is essential to support the long-term cell motility in a wound bed, possibly by inhibition of plasmin generation [33, 34]. Research focusing on the mechanism of cancer metastasis have proven that serpin E1 exhibits an inhibitory activity against MMPs [35]. Therefore, the presence of serpin E1 in a wound bed may reveal beneficial effects on wound healing process. The analysis using protein microarray has shown the presence of comparable amounts of serpin E1 in amnion samples derived from physiological deliveries, and cesarean sections. The median level of serpin E1 was also similar in both placental and cervical parts of membranes in both groups.
The present study is a preliminary screening analysis, thus performed on a limited number of samples. Based on the obtained results, it would be now possible to assess the identified biologically active factors in a larger group of AM, especially using the quantitative methods, such as ELISA assessment. Nevertheless, our proof-of-concept study has shown that the content of biologically active factors in AM samples is highly variable, mainly due to inter-donor variations. Moreover, it depends on the specific area of the amniotic membrane, and the mode of childbirth. Despite the small number of AM samples included in the study, the results of our proof-of concept study may explain inconsistent results of clinical research.
The presence of several biologically active factors, which regulate tissue regeneration, makes the amniotic membrane a valuable candidate for biological dressing that actively stimulates process of wound healing. Previous studies have shown that the content of these factors in AM samples and, thus, their biological properties may depend on various methods of AM processing and sterilization [11, 12]. However, results of our proof-of-concept study suggest that properties of AM-derived dressing samples also depend on the part of harvested AM sample, as well as on the delivery method. This concept has been already postulated by Banerjee et al., who have observed the higher mitochondrial respiratory activity of AM samples derived from placental region, in comparison to reflected (distant from placenta) region of the membrane [36]. Our observation suggests that placental region of AM, especially in membranes from physiological delivery, is characterized by high concentrations of growth factors and regulators of ECM turnover that stimulate process of tissue regeneration. Therefore, it may be more suitable for application in a treatment of non-healing wounds. On the other hand, amniotic membranes obtained from elective cesarean section, especially their cervical portions, are relatively poor in pro-angiogenic factors. These characteristics could be potentially beneficial e.g. for ophthalmological applications, like closure of corneal lesions and ocular surface reconstruction, where stimulation of fibroblasts proliferation or angiogenesis is not desired. However, to verify our observations, further research with larger group is necessary.

Authors declare no conflict of interest.

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