Amniotic Membrane Scaffold Research Articles

Amniotic Membrane Scaffolds Enable the Development of Tissue-Engineered Urothelium with Molecular and Ultrastructural Properties Comparable to that of Native Urothelium

The amniotic membrane (AM) is a naturally derived biomaterial that possesses biological and mechanical properties of great importance for tissue engineering. The aim of our study was to determine whether the AM enables the formation of a normal urinary bladder epithelium-urothelium--and to reveal any differences in the urothelial cell (UC) growth and differentiation when using different AM scaffolds. Cryopreserved human AM was used as a scaffold in three different ways. Normal porcine UCs were seeded on the AM epithelium (eAM), denuded AM (dAM), and stromal AM (sAM) and were cultured for 3 weeks. UC growth on AM scaffolds was monitored daily. By using electron microscopy, histochemical and immunofluorescence techniques, we here provide evidence that all three AM scaffolds enable the development of the urothelium. The fastest growth and the highest differentiation of UCs were demonstrated on the sAM scaffold, which enables the development of tissue-engineered urothelium with molecular and ultrastructural properties comparable to that of the native urothelium. Most importantly, the highly differentiated urothelia on the sAM scaffolds provide important experimental models for future drug delivery studies and developing tissue engineering strategies considering that subtle differences are identified before translation to the clinical settings.

Acellular amniotic membrane: an appropriate scaffold for fibroblast proliferation

Various materials have been investigated as possible skin substitutes to repair skin defects such as burns. Because of its unique characteristics, acellular amniotic membrane seems to provide a good scaffold for cell cultures. To investigate the proliferation of fibroblasts on an amniotic membrane scaffold, and the preparation of a temporary skin substitute using this method. Neonatal foreskin tissue was harvested after circumcision and used for isolation of skin fibroblasts. The skin sample was refrigerated in cell-culture solution, and later treated with trypsin, minced, and incubated in the same solution at 37 °C with in an atmosphere of 95% O2 /5% CO2 . The confluent cultures were treated with trypsin, and fibroblasts were subcultured up to the 10th passage. Cells were tested for microbial contamination, presentation of major histocompatibility complex, and karyotype changes. Amniotic membrane was harvested after elective caesarean section from donors who had been screened for infection. The membrane was washed and then subjected to three freeze-thaw cycles, before having the cells removed. The fibroblasts were seeded onto the scaffold, and after 24 h, the prepared skin substitute was ready. This was examined under electron microscopy. The skin substitute showed excellent growth of fibroblasts on the amniotic membrane scaffold. Fibroblasts had excellent adherence to and viability on the acellular amniotic membrane, which seems to provide an acceptable temporary skin substitute that can be used for wounds.

Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model

BackgroundThe early peritoneal invasion of epithelial ovarian cancer (EOC) by tumoral aggregates presents in ascites is a major concern. The role of the microenvironment seems to be important in this process but the lack of adequate models to study cellular interactions between cancer cells and stromal cells does not allow to uncover the molecular pathways involved. Our goal was to study the interactions between ovarian cancer cells (OCC) and mesenchymal stem cells (MSC) using a 3D model.MethodsWe used millimetric pieces of amniochorionic membrane - referred to as amniotic membrane scaffold (AMS) - to create 3D peritoneal nodules mimicking EOC early invasion. We were able to measure the distribution and the depth of infiltration using confocal microsopy. We extracted MSC from the amniochorionic membrane using the markers CD34-, CD45-, CD73+, CD90+, CD105+ and CD29+ at the Fluorescence Activated Cell Sorting (FACS) analysis. We used transwell and wound healing tests to test OCC migration and invasion in vitro.ResultsHere we show that OCC tumors were located in regions rich in MSC (70%). The tumors infiltrated deeper within AMS in regions rich in MSC (p<0.001). In vitro tests revealed that higher IL6 secretion in a context of MSC-OCC co-culture could enhance migration and invasion of OCC. After IL6 receptor antagonism, OCC infiltration was significantly decreased, mostly in regions rich in MSCs, indicating that recruitment and tridimensional invasion of OCC was dependent of IL6 secretion.ConclusionsThe use of tridimensional models using AMS could be a useful tool to decipher early molecular events in ovarian cancer metastasis. Cytokine inhibitors interrupting the cross-talk between OCCs and MSCs such as IL6 should be investigated as a new therapeutic approach in ovarian cancer.

The impact of compositional topography of amniotic membrane scaffold on tissue morphogenesis of salivary gland

Amniotic membrane (AM) has been widely used in the reconstruction of oral epithelial defects. However, whether it is also effective in facilitating tissue formation of salivary gland, an appendix of oral epithelia, has never been explored. To investigate the effects and the underlying mechanism of AM on salivary gland morphogenesis, murine fetal submandibular gland (SMG) explants were cultured on different preparations of AM scaffolds. It was found that, on AM stromal scaffold, SMG demonstrated well-developed branching morphogenesis. Nonetheless, on AM epithelial scaffold, SMG epithelial cell converted to a spindle-shape, lost intercellular connection, changed cytoskeletal organization, and exhibited scattering behaviors. Meanwhile, the integrity of SMG basement membrane was dismantled as well. However, when acellular AM epithelial scaffold was used, cultured SMG demonstrated organized morphology, indicating that AM epithelial component provided specific surface features for SMG morphogenesis. To further investigate AM scaffold morphogenetic effect, it was found hepatocyte growth factor (HGF), an epithelial scattering factor, was expressed abundantly in cultivated AM epithelia. After blocking HGF function of AM, cultured SMG regained branching activity, reorganized cell adhesion and subcellular organization, and reproduced basement membranes. Therefore, AM-derived bioactive factor profoundly influences cell behaviors and structure formation of SMG. Together, this study showed that compositional topography of AM scaffold is important in affecting SMG morphogenesis. By understanding the effects of AM scaffold on SMG morphogenesis, it provides important information for rationally designing and fabricating AM scaffold for salivary gland regeneration.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

To compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. Native AS were engineered from freshly harvested membranes of 23d gestational age (GA; term=31-2d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA=23d and primarily closed with AS (n=10) or DegraPol(n=10) or left unclosed (positive controls; n=10). Sixty-one sacs served as negative controls. At GA=30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. Engineered AS had a cell-free collagenous fiber network. AF tended to be, or was higher in the AS (67%; P=.07) or DegraPol group (78%; P<.05) as compared to positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher re-epithelialization rates (AS: 52.5%±6.5%; DegraPol: 11.6%±2.6; P<.001) and proliferation indices (AS: 0.47±0.03, DegraPol: 0.28±0.04; P=.001). In both treatment groups, cell proliferation in the myometrium was increased (P<.05). Native AS seal iatrogenic fetal membrane defect better than DegraPol. Within a week, there is abundant re-epithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.