Safety and clinical efficacy of the secretome of stressed peripheral blood mononuclear cells in patients with diabetic foot ulcer-study protocol of the randomized, placebo-controlled, double-blind, multicenter, international phase II clinical trial MARSYAS II.

BACKGROUND: Diabetes and its sequelae such as diabetic foot ulcer are rising health hazards not only in western countries but all over the world. Effective, yet safe treatments are desperately sought for by physicians, healthcare providers, and of course patients. METHODS/DESIGN: APOSEC, a novel, innovative drug, is tested in the phase I/II study MARSYAS II, where its efficacy to promote healing of diabetic foot ulcers will be determined. To this end, the cell-free secretome of peripheral blood mononuclear cells (APOSEC) blended with a hydrogel will be applied topically three times weekly for 4 weeks. APOSEC is predominantly effective in hypoxia-induced tissue damages by modulating the immune system and enhancing angiogenesis, whereby its anti-microbial ability and neuro-regenerative capacity will exert further positive effects. In total, 132 patients will be enrolled in the multicenter, randomized, double-blind, placebo-controlled, parallel group, dose-ranging phase I/II study and treated with APOSEC at three dose levels or placebo for 4 weeks, followed by an 8-week follow-up period to evaluate safety and efficacy of the drug. Wound area reduction after 4 weeks of treatment will serve as the primary endpoint. CONCLUSION: We consider our study protocol to be suitable to test topically administered APOSEC in patients suffering from diabetic foot ulcers in a clinical phase I/II trial. TRIAL REGISTRATION: EudraCT 2018-001653-27 . Registered on 30 July 2019. ClinicalTrials.gov NCT04277598 . Registered on 20 February 2020. TITLE: "A randomized, placebo-controlled, double-blind study to evaluate safety and dose-dependent clinical efficacy of APO-2 at three different doses in patients with diabetic foot ulcer (MARSYAS II)".

Adipose-derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix.

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system.

Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.

Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model.

The human amniotic membrane (hAM), thanks to its favorable properties, including anti-inflammatory, anti-fibrotic and pro-regenerative effects, is a well-known surgical material for many clinical applications, when used both freshly after isolation and after preservation. We have shown previously that hAM patching is a potential approach to counteract liver fibrosis. Indeed, when fresh hAM was used to cover the liver surface of rats with liver fibrosis induced by the bile duct ligation (BDL) procedure, the progression and severity of fibrosis were significantly reduced. Since cryopreservation enables safety and long-term storage of hAM but may influence its functional properties, here we compared the anti-fibrotic effects of fresh and cryopreserved hAM in rats with BDL-induced liver fibrosis. After BDL, the rat liver was covered with a piece of fresh or cryopreserved hAM, or left untreated. Six weeks later, the degree of liver fibrosis was assessed histologically using the Knodell and the METAVIR scoring systems. Digital image analysis was used to quantify the percentage of the areas of each liver section displaying ductular reaction, extracellular matrix (ECM) deposition, activated myofibroblasts and hepatic stellate cells (HSCs). Liver collagen content was also determined by spectrophotometric technique. The degree of liver fibrosis, ductular reaction, ECM deposition, and the number of activated myofibroblasts and HSCs were all significantly reduced in hAM-treated rats compared to control animals. Fresh and cryopreserved hAM produced the same anti-fibrotic effects. These findings indicate that cryopreservation maintains the anti-fibrotic properties of hAM when used as a patch to reduce the severity of liver fibrosis.

Development and validation of a production process of platelet lysate for autologous use.

Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation.

Mesenchymal stem cells (MSCs) possess immunosuppressive properties. But also fully differentiated human renal tubular epithelial cells (RTECs) are able to modulate T-cell proliferation in vitro. In this study we compared two MSC populations, human adipose derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs), and RTECs regarding their potential to inhibit monocyte-derived dendritic cell (DC) differentiation and maturation in indirect co-culture. In the presence of hAMSCs and RTECs, monocytes stimulated to undergo DC differentiation were inhibited to acquire surface phenotype of immature and mature DCs. In contrast, ASCs showed only limited suppressive capacity. Secretion of IL-12p70 was suppressed in hAMSC co-cultures and high IL-10 levels were detected in all co-cultures. Prostaglandin E(2) was found in ASC and hAMSC co-cultures, whereas soluble human leukocyte antigen-G was highly elevated only in RTEC co-cultures. Thus, inhibition of DC generation by MSCs and RTECs might be mediated by different soluble factors.

Osteogenic differentiation of intact human amniotic membrane.

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.

Alternative sources of adult stem cells: human amniotic membrane.

Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.