Standardized Human Platelet Lysates as Adequate Substitute to Fetal Calf Serum in Endothelial Cell Culture for Tissue Engineering.

Fetal calf serum (FCS) is used for in vitro cell culture, as it provides the cells with various growth-promoting compounds. For applications in humans, FCS does not meet the required safety standards and should be replaced by an appropriate substitute. This study analyzed the suitability of using human platelet lysate (hPL) as a substitute for FCS in endothelial cell cultures for in vitro and in vivo tissue engineering applications. The focus was placed on standardized, commercially available hPLs (MultiPL'30, MultiPL'100), which are approved for applications in humans, and compared to laboratory-prepared hPLs (lp-hLP). Human umbilical vein endothelial cells (HUVEC) were cultured with FCS or with different hPLs. Cell morphology, proliferation, viability, apoptosis, and necrosis, as well as the organization of vascular structures, were assessed. No morphological changes were noticed when FCS was replaced by standardized hPLs in concentrations of 1-10%. In contrast, the use of lp-hLPs led to irregular cell shape and increased vacuolization of the cytoplasm. HUVEC proliferation and viability were not compromised by using media supplemented with standardized hPLs or pl-hPLs in concentrations of 1-10%, compared to cells grown in media supplemented with 20% FCS. The apoptosis rate using lp-hPLs was higher compared to the use of standardized hPLs. The necrosis rate tended to be lower when FCS was replaced by hPLs. HUVEC formed more pronounced capillary-like structures when the media were supplemented with hPLs instead of supplementation with FCS. Thus, compared to the use of FCS, the use of hPLs was beneficial for the growth and optimal expression of functional endothelial cell characteristics during in vitro experiments. Commercially available hPLs proved to be particularly suitable, as they led to reproducible results during in vitro experiments, while meeting the safety requirements for in vivo use.

Full-thickness tissue engineered oral mucosa for genitourinary reconstruction: A comparison of different collagen-based biodegradable membranes.

Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.

The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents.

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents.

Risk Factors for Chemotherapy-Associated Venous Thromboses in Gynaecological Oncology Patients.

Introduction Venous thromboses and their consequences are among the main causes of death in patients with tumour diseases. The objective of this study is the analysis of risk factors and the evaluation of the applicability of two risk scores in a purely gynaecological oncology patient collective. The identification of patients at high risk for the occurrence of venous thromboses could enable the implementation of targeted medication-based thrombosis prophylaxis which has a significant benefit and, simultaneously, a low risk. Materials and Methods A retrospective case-control study on 152 patients who were undergoing oncological treatment in the Department of Gynaecology of the Mainz University Medical Centre between 2006 and 2013 investigated the data from 104 patients with breast, 26 with ovarian and 22 with cervical cancer. A control was assigned to 76 subjects in the case group who suffered a venous thrombosis during chemotherapy and this control coincided in the points of tumour location, age, lymph node involvement, metastasis and time of initial diagnosis. The group differences were analysed using the χ 2 test, t test, Mann-Whitney-U test and a logistic regression analysis. Results There were clear group differences in the lack of inpatient thrombosis prophylaxis (p = 0.014), elevated leukocyte counts (p = 0.018) prior to the start of chemotherapy and port systems (p = 0.032). Surgical interventions were confirmed to be an independent risk factor (p ≤ 0.001). The Khorana and Protecht scores did not emerge from the analysis as independent predictors for a thrombosis. More patients died in the case group than in the control group (p = 0.028; OR: 8.1; CI: 1.254 - 52.162). Conclusion In this patient collective, surgeries represent an independent risk factor for venous thromboses. In addition, a correlation was seen between inpatient thrombosis prophylaxis, leukocytosis as well as port systems and an increased risk of thrombosis. Neither the Khorana nor the Protecht score were independent risk factors for venous thromboses. Significantly more thrombosis patients died during the observation period.

Primary Mucosal Epithelial Cell Cultivation: A Reliable and Accelerated Isolation.

IMPACT STATEMENT: We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevertheless reliable method.