Manufacture of a Cell-free Amnion Matrix Scaffold that Supports Amnion Cell Outgrowth In Vitro

Abstract

We manufactured a cell-free extracellular matrix scaffolds in order to obtain a support material for amnion cell outgrowth, eventually being used for repair of prematurely ruptured fetal membrane. Human preterm or term amnion tissue was separated into its collagenous extracellular matrix and cell components. The acellular scaffold was explored for its capacity to support regrowth of isolated human amnion epithelial or mesenchymal cells in vitro. The outgrowth of amnion cells on and in the scaffold was investigated by scanning and transmission electron microscopy, and confocal laser scanning microscopy.

Cell-free amnion matrix scaffolds demonstrated a porous collagen fiber network similar as in native amnion. Inoculation of acellular amnion scaffolds with human amnion cells revealed that its property to support amnion cell outgrowth was retained. Amnion epithelial and mesenchymal cells were found to grow into dense layers on the surface of the scaffold within 3–4 days and 7–8 days, respectively, and to some extent, invaded the scaffold during the culture period.

Manufactured acellular amnion matrix retains structural and functional properties required for cell outgrowth in vitro. It may become useful to repair prematurely ruptured fetal membranes.

Introduction

The human fetal membranes surrounding the amniotic cavity are composed of the amnion and the chorion [1]. The amnion is the interior part consisting of a single layer of epithelial cells (human amnion epithelial cells, HAECs) on a thicker basement membrane and collagen spongy layer containing mesenchymal cells (human amnion mesenchymal cells, HAMCs). It retains the amniotic fluid, secretes mediators such as prostaglandins, cytokines and protein hormones both into the amniotic fluid and towards the uterus, and guards the fetus against infections ascending the genital tract and from mechanical injury [2].

Spontaneous preterm premature rupture of the amniotic membranes (sPPROM), at less than 37 completed gestational weeks, occurs in 1–2% of all pregnancies and accounts for 30–40% of all preterm deliveries [2], [3].

The etiology of sPPROM is multifactorial [2], [4] and includes, for example, mechanical stress, infection or cervical incompetence [2], [4]. In addition, we are confronted with cases of iatrogenic PPROM (iPPROM), a potential complication and limiting step of invasive prenatal procedures. The incidence is 1.2% after genetic amniocentesis [5], 3–5% after diagnostic fetoscopy [6], >30% after fetoscopic cord ligation [7] and >62% after endoscopic tracheal clipping [8]. Although there is a classification in spontaneous and iatrogenic PPROM, the overall perinatal mortality of previable PPROM managed expectantly is reported to be between 55% and 66% [9], [10]. Actual management differs between immediate delivery or termination of pregnancy and conservative management with or without the use of tocolytics, antibiotics, and steroids in various combinations [3].

Current research is focused on finding methods to anatomically and functionally restore ruptured membranes. Approaches include the amniopatch [11], [12], [13], amniograft [14], intracervical fibrin tissue sealants [15], [16], gelatin sponge plugs [17], [18], collagen plugs [19], [20] and human amnion pieces [20]. Although these attempts possess apparent potential, they are small case-based studies and it is difficult to apply these generally. The search for alternative methods, such as in this study, is mandatory.

The aim of the present study was to explore the utility of both cellular as well as manufactured extracellular components of human amnion for reconstituting defective amnion in PPROM patients, using the methods of tissue engineering (Fig. 1).