Tissue engineering for neurodegenerative diseases using human amniotic membrane and umbilical cord.

Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton's jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4-5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.

Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent.

Human amniotic membrane (HAM) has useful properties as a dermal matrix substitute. The objective of our work was to obtain, using different enzymatic or chemical treatments to eliminate cells, a scaffold of acellular HAM for later use as a support for the development of a skin equivalent. The HAM was separated from the chorion, incubated and cryopreserved. The membrane underwent different enzymatic and chemical treatments to eliminate the cells. Fibroblasts and keratinocytes were separately obtained from skin biopsies of patients following a sequential double digestion with first collagenase and then trypsin-EDTA (T/E). A skin equivalent was then constructed by seeding keratinocytes on the epithelial side and fibroblasts on the chorionic side of the decellularizated HAM. Histological, immunohistochemical, inmunofluorescent and molecular biology studies were performed. Treatment with 1% T/E at 37 °C for 30 min totally removed epithelial and mesenchymal cells. The HAM thus treated proved to be a good matrix to support adherence of cells and allowed the achievement of an integral and intact scaffold for development of a skin equivalent, which could be useful as a skin substitute for clinical use.

Anatomopathological and immunohistochemical study of explanted cryopreserved arteries.

BACKGROUND: The aim of the study was to analyze the mechanism of deterioration of implanted arteries. METHODS: Eleven patients were included. Samples of vascular segments obtained from multiorgan donors and samples of the same vascular segments after explantation in the recipient were analyzed. Blood group, time of cold and warm ischemia, cause of death, time spent in the intensive care unit, time of storage of the cryopreserved grafts, and anatomopathological and immunohistochemical studies were analyzed using the preimplant samples obtained from the multiorgan donor. For samples obtained from the recipient, blood group, duration for which the tissue from the donor has been implanted, reason for graft explantation, and anatomopathological and immunohistochemical studies were analyzed. RESULTS: Histopathologically, the main finding has been the substitution of the muscular cap of the arterial wall by an intense fibrosis, in most of the cases, of a symmetrical nature. Besides this degeneration of myocytes, there is marked perivascular fibrosis and fibrointimal thickening also exists. The T lymphocytes suggest the importance of the immunological mechanism in the distortion of the architecture of the arteries. The atherosclerosis plays a less relevant role. CONCLUSIONS: Evidence of immune-mediated injury was found, and this mechanism seems to be responsible for the degenerative process in cryopreserved homografts.

Multilineage differentiation potential of cells isolated from the human amniotic membrane.

The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM-derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion-derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem-cell markers SSEA-4 and STRO-1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy.

Potential use of the human amniotic membrane as a scaffold in human articular cartilage repair.

The human amniotic membrane (HAM) is an abundant and readily obtained tissue that may be an important source of scaffold for transplanted chondrocytes in cartilage regeneration in vivo. To evaluate the potential use of cryopreserved HAMs as a support system for human chondrocytes in human articular cartilage repair. Chondrocytes were isolated from human articular cartilage, cultured and grown on the chorionic basement membrane side of HAMs. HAMs with chondrocytes were then used in 44 in vitro human osteoarthritis cartilage repair trials. Repair was evaluated at 4, 8 and 16 weeks by histological analysis. Chondrocytes cultured on the HAM revealed that cells grew on the chorionic basement membrane layer, but not on the epithelial side. Chondrocytes grown on the chorionic side of the HAM express type II collagen but not type I, indicating that after being in culture for 3-4 weeks they had not de-differentiated into fibroblasts. In vitro repair experiments showed formation on OA cartilage of new tissue expressing type II collagen. Integration of the new tissue with OA cartilage was excellent. The results indicate that cryopreserved HAMs can be used to support chondrocyte proliferation for transplantation therapy to repair OA cartilage.

Functional assessment of cryopreserved human femoral arteries for pharmaceutical research.

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings). Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs. After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered. The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.

Functional assessment of cryopreserved pig aortas for pharmaceutical research.

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached. After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.

Functional assessment of cryopreserved human aortas for pharmaceutical research.

We evaluated the impact of standard cryopreservation on functional properties of human aortic homografts. From seven human donors, the thoracic descending aorta was obtained. Effects of cryopreservation on contractibility and endothelium function were tested. After cryopreservation no endothelium-dependent or endothelium-independent relaxation was found and the contractibility was strongly affected. Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing.