Aims. The human amniotic membrane (hAM), used in regenerative medicine, contains stem cells. For clinical application, profound knowledge of properties of applied materials is a prerequisite. In ischemic rat heart, hAM patches reduced cardiac dysfunctiona, a pathologic event associated with mitochondrial dysfunction. Studies have shown that mitochondrial transfer can protect against, for example, acute lung injuryb. Therefore, we investigated whether sub-regions of hAM (placental-P and reflected-R) show different mitochondrial function, mtDNA contents and reactive oxygen species (ROS) levels. Methods. Mitochondrial respiration was monitored by high-resolution respirometry. ROS production was measured with electron paramagnetic resonance spectrometry. Results. Amniotic sub-regions differ in activity, as P has significantly higher mitochondrial respiration, however significantly less ROS. Respiratory control ratio showed no differences, reflecting similar mitochondrial quality in both regions, suggesting higher numbers of mitochondria in P. MtDNA analysis showed similar copy numbers per cell in both regions indicating similar cellular mitochondrial density, and hence higher cellularity of P. Conclusion. The amniotic sub-regions, show distinct differences in mitochondrial activity and ROS production. Consequently, the amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.

Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.

Human amniotic membrane (hAM) represents a tissue that is well established as biomaterial in the clinics with potential for new applications in regenerative medicine. For tissue engineering (TE) strategies, cells are usually combined with inductive factors and a carrier substrate. We have previously recognized that hAM represents a natural, preformed sheet including highly potent stem cells. In the present approach for cartilage regeneration we have induced chondrogenesis in hAM in vitro. For this, hAM biopsies were cultured for up to 56 days under chondrogenic conditions. The induced hAM was characterized for remaining viability, glycosaminoglycan (GAG) accumulation using histochemical analysis, and a quantitative assay. Collagen I, II and X was immunohistochemically determined and cartilage-specific mRNA expression of (sex determining region Y-) box 9, cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA), and cartilage-linking protein 1 (CRTL1) analyzed by quantitative real-time polymerase chain reaction. Human AM was successfully induced to accumulate GAG, as demonstrated by Alcianblue staining and a significant (p < 0.001) increase of GAG/viability under chondrogenic conditions peaking in a 29.9 ± 0.9-fold induction on day 56. Further, upon chondrogenic induction collagen II positive areas were identified within histological sections and cartilage-specific markers including COMP, AGC1, CSPG2, COL1A1, COL9A2, MIA, and CRTL1 were found upregulated at mRNA level. This is the first study, demonstrating that upon in vitro induction viable human amnion expresses cartilage-specific markers and accumulates GAGs within the biomatrix. This is a promising first step towards a potential use of living hAM for cartilage TE.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.

Farmaco para uso local para fomentar la regeneracion de tejidos, caracterizado en que - contiene microparticulas que derivan de trombocitos y son obtenibles mediante un tratamiento activadorde los trombocitos con trombina, colageno, el ionoforo de Ca2+ A23187 y/o la proteina C5b-9 en solucionacuosa y se han purificado mediante centrifugacion diferencial, filtracion o cromatografia de afinidad, - se han sometido a un procedimiento de inactivacion de virus y/o disminucion de virus, - y que se alcanzo la esterilidad mediante filtracion esteril, y que - esta en un estado liofilizado o ultracongelado.

To evaluate the effects of anticoagulant agent (low-molecular-weight heparin, LMWH) on the pulmonary artery intima hyperplasia of rats with acute pulmonary embolism (APE) by assaying platelet-derived growth factor-β (PDGF-β).A total of 90 Sprague-Dawley rats were randomly assigned into the sham, APE, and LMWH groups with 30 rats in each group. The APE rat models were established by injecting autologous blood clots via external jugular veins. In each group, six mice were sacrificed at the 1st day (D1), 4th day (D4), 7th day (D7), 14th day (D14), and 28th (D28) subsequent to the induction of APE to collect the lungs. Right ventricle pressure (RVP) and mean pulmonary arterial pressure (mPAP) were measured. Western blot and RT-PCR analyses were used to assess PDGF-β expression at various time points. In addition, changes in lung pathology were evaluated using hematoxylin and eosin (H&E) staining and electron microscope.The overall success rate of establishing APE rat models was 85.7% (60/70). There was no difference in mPAP between the sham group and the APE group at the D1, D4, D7, and D14. However, at the D28, mPAP in the APE group was significantly higher than that in the sham group. There was no difference among the three groups regarding RVP. PDGF-β expression were decreased in the LMWH group at all time points compared with the sham and APE groups (P < 0.01). Furthermore, pulmonary embolism, alveolar wall necrosis and hemorrhage, and inflammation were significantly attenuated in the LMWH group compared with the sham and APE groups subsequent to the induction of APE.LMWH attenuates lung and pulmonary artery injuries and improves prognosis. Decreased PDGF-β in the lungs may be the important factor in the effects observed.

Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. In our approach, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials. For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM viability was evaluated. Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4 ± 6.8 mg/dl d28), increased AP activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3 ± 19.0% d14; 62.9 ± 22.3% d28) compared to CM (42.2 ± 15.2% d14; 35.1 ± 8.6% d28). By this strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment.

Galway, Ireland Osteogenic Differentiation of Human Amniotic Membrane in toto Andrea Lindenmair, Susanne Wolbank, Anja Peterbauer-Scherb, Guido Stadler, Christian Gabriel, Johann Eibl, Martijn van Griensven, Heinz Redl, Corresponding Author: susanne.wolbank@trauma.lbg.ac.at Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center Linz/Vienna, Austria and Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria and Cell Biology, UT Southwestern Medical Center, Dallas, Texas, USA and Bio-Products & Bio-Engineering AG, Vienna, Austria and Austrian Cluster for Tissue Regeneration, Vienna, Austria

Currently freeze-dried, gamma-sterilized, or glycerol-preserved amniotic membranes are widely used. However, it is not clear whether this devitalized state is the optimal application form. Therefore within this study the ideal condition for midterm storage of human amniotic membranes was assessed to ensure the availability of vital amniotic membranes, in particular for burn wounds. For this purpose mothers were serologically tested and term placentae were collected and washed. After the amniotic membrane was peeled off and further washed, biopsies were taken for microbiological testing and various storage experiments (different media and temperatures). cell culture medium, 37 °C glycerol, 4 °C 10% DMSO, −80 °C Viability of fresh and stored amniotic membranes was determined with the MTT-based EZ4U- Assay (Biomedica, Vienna, Austria). Best results were obtained while storing the membranes in cell culture medium at 37 °C, whereas storage in glycerol at 4 °C resulted in cell death within the first 4 days. To our knowledge this is the first study investigating the viability of amniotic membrane under different storage conditions. The influence on wound healing is currently under investigation.