Impact of Porcine Pancreas Decellularization Conditions on the Quality of Obtained dECM.

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.

The Influence of the Flow of Detergent and Donor Characteristics on the Extracellular Matrix Composition After Human Pancreas Decellularization.

INTRODUCTION: The extracellular matrix (ECM) consists, among others, of polysaccharides, glycosaminoglycans, and proteins. It is being increasingly used in tissue bioengineering. Obtaining ECM of the highest quality through decellularization is a big challenge because of some differences in organ structure. To deprive organs of the cellular part, chemical, enzymatic, or mechanical methods are used. After decellularization, we get a scaffold made of a variety of proteins, and it is the role of these proteins that can significantly affect the maintenance of the spatial structure and be a suitable environment for cells to rebuild a specific organ. AIM: Estimation of the detergent (Triton X-100) flow parameters and anthropometric donors' decellularization process accuracy on the final ECM composition. MATERIALS: Five human pancreata, rejected from transplantation, were used for decellularization. All organs were harvested from brain-dead donors age 13 to 60 years. METHODS: Decellularization was carried out using the flow method with Triton X-100 as an active agent. The experiment compared 5 different flow values. After decellularization, an assessment of the final DNA concentration and the protein composition was performed. Results were compared to anthropometric data of donors. In addition, a microscopic analysis was also carried out. RESULTS: The best results were obtained using a flow of 120 mL/minute. A higher detergent flow was associated with a lower concentration of residual DNA in scaffold. Analysis of the protein profile with anthropometric data has shown that LAM A2 was increasing with age and LAMA5 was decreasing. Being overweight was associated with a higher proportion of COL1 and 4 and a smaller proportion of COL6.

Three-dimensional nanofibrous polystyrene scaffolds modify macrophage phenotypes and activate macrophage angiogenic potential.

3D scaffolds represent an attractive substrate for studying macrophage activation and modification since they mimic extracellular matrix (ECM). However, macrophage response to such materials, particularly with respect to angiogenic potential is still poorly recognized. Therefore, we investigated the effect of 3D nanofibrous polystyrene scaffolds (NPSs) versus tissue culture polystyrene (TCPS) on THP-1-derived macrophages in various environmental conditions, for example, standard (m0), pro-inflammatory (m1), or anti-inflammatory (m2) with respect to pro-angiogenic potential. There were no differences in the expression of TNF-α and IL-10 mRNAs and respective proteins in cells cultured on NPSs compared with flat polystyrene (TCPS), however, NPSs induced an increased VEGF production by macrophages cultured in m0 and m1 media. Cells cultured in m1, and m2 conditions secreted elevated amounts of TNF-α and IL-10, respectively, irrespective of substrate surface geometry. Each macrophage population contains large, medium, and small cells. Moreover, there were significant differences in the proportion of large to small macrophages depending on the medium composition, that is, in m0, m1, and m2 media these proportions were 1:4, 1:3, and 1:10, respectively. The ultrastructure and the immunoexpression of TNF-α and IL-10 were analyzed under a confocal microscope. The results demonstrated differences in cell ultrastructure and suggested that the larger cells were pro-inflammatory macrophages, while the smaller cells were anti-inflammatory macrophages. In conclusion, NPSs activate macrophage pro-angiogenic potential. In addition, an increase in the proportion of pro-inflammatory macrophages relative to anti-inflammatory ones in a given population favors this potential.

How calcite and modified hydroxyapatite influence physicochemical properties and cytocompatibility of alpha-TCP based bone cements.

Nowadays successful regeneration of damaged bone tissue is a major problem of the reconstructive medicine and tissue engineering. Recently a great deal of attention has been focused on calcium phosphate cements (CPCs) as the effective bone fillers. Despite a number of studies regarding CPCs, only a few compare the physicochemical and biological properties of α-TCP based materials of various phase compositions. In our study we compared the effect of several components (calcite, hydroxyapatite doped with Mg2+, CO32- or Ag+ ions, alginate, chitosan and methylcellulose) on the physicochemical and biological properties of α-TCP-based bone cements. The influence of materials composition on their setting times, microstructure and biochemical stability in simulated body fluid was determined. A number of in vitro laboratory methods, including ICP-OES, metabolic activity test, time-lapse microscopic observation and SEM observations were performed in order to assess biocompatibility of the studied biomaterials. The positive outcome of XTT tests for ceramic extracts demonstrated that all investigated cement-type composites may be considered cytocompatible according to ISO 10993-5 standard. Results of our research indicate that multiphase cements containing MgCHA, AgHA and calcite combined with αTCP enhanced cell viability in comparison to material based only on αTCP. Furthermore materials containing chitosan and methylcellulose possessed higher cytocompatibility than those with alginate.

Vasculogenesis and Its Cellular Therapeutic Applications.

Vasculogenesis was originally defined by Risau in 1997 [Nature 386: 671-674] as the de novo formation of vessels from endothelial progenitor cells (EPCs), so-called angioblasts. Initially, this process was believed to be related only to embryonic life; however, further studies reported vasculogenesis to occur also in adult tissues. This overview presents the current knowledge about the origin, differentiation and significance of EPCs that have been observed in various diseases, tumors, and reparative processes. We also summarize the knowledge of how to activate these cells for therapeutic purposes and the outcomes of the therapies.

Cytocompatibility of the selected calcium phosphate based bone cements: comparative study in human cell culture.

Calcium phosphate cements (CPC) are valuable bone fillers. Recently they have been also considered as the basis for drug-, growth factors- or cells-delivery systems. Broad possibilities to manipulate CPC composition provide a unique opportunity to obtain materials with a wide range of physicochemical properties. In this study we show that CPC composition significantly influences cell response. Human bone derived cells were exposed to the several well-characterized different cements based on calcium phosphates, magnesium phosphates and calcium sulfate hemihydrate (CSH). Cell viability assays, live/dead staining and real-time observation of cells in contact with the materials (time-laps) were performed. Although all the investigated materials have successfully passed a standard cytocompatibility assay, cell behavior in a direct contact with the materials varied depending on the material and the experimental system. The most recommended were the α-TCP-based materials which proved suitable as a support for cells in a direct contact. The materials which caused a decrease of calcium ions concentration in culture induced the negative cell response, however this effect might be expected efficiently compensated in vivo. All the materials consisting of CSH had negative impact on the cells. The obtained results strongly support running series of cytocompatibility studies for preclinical evaluation of bone cements.

Development of a pre-vascularized 3D scaffold-hydrogel composite graft using an arterio-venous loop for tissue engineering applications.

Hyaluronic acid (HA) and fibrin glue (FG) are effective hydrogels for tissue engineering applications as they support tissue in-growth, retain growth factors, and release them slowly with time. The scaffolds, in combination with a hydrogel, effectuate a successful graft. However, the survival of a graft entirely depends upon a functional vascular supply. Therefore, hydrogels must support the in-growing vasculature. To study and compare the vascular patterns, HA and FG hydrogel-containing PLDLLA-TCP-PCL scaffolds were implanted in the groin of male Lewis rats and supplied with a micro-surgically prepared arterio-venous (A-V) loop. The rats were perfused with a vascular contrast media after 4 and 8 weeks and sacrificed for further analysis. The specimens were scanned with micro-CT to find the vascular growth patterns. Corrosion casting of blood vessels followed by SEM demonstrated a high vascular density near the parent blood vessels. Histologically, HA and FG implanted animal groups showed significant angiogenetic activity, especially within the pores of the scaffold. However, formation of new blood vessels was more conspicuously observed at 4 weeks in FG than HA implants. Furthermore, by 8 weeks, the number and pattern of blood vessels were comparable between them. At this time, HA was still present indicating its slow degradation. The finding was confirmed by histomorphometric analysis. This experimental study demonstrates that HA containing composite scaffold systems permit stabile in-growth of blood vessels due to sustained degradation over 8 weeks. HA is a potential matrix for a tissue engineered composite graft.

Poly(epsilon-caprolactone) biomaterial sterilized by E-beam irradiation.

The effects of ionizing radiation (electron beam) on poly(epsilon-caprolactone) (PCL) were studied by analyzing changes in viscosity-average and weight-average molecular weight and radius of gyration, and by performing sol-gel analysis and swelling tests. Samples were irradiated under various conditions: solid and molten PCL in the presence or absence of air. The overall efficiency of crosslinking is higher for samples irradiated in the molten state than in the solid state, and is reduced in the presence of oxygen. Based on three kinds of experiments (molecular weight dependence on the dose in the pre-gelation region, sol-gel analysis, and swelling study), radiation-chemical yields of intermolecular crosslinking and scission were determined and are discussed in terms of the mechanism of radiation-induced reactions in PCL. Properties of the gels formed by high-dose irradiation and mechanical properties of irradiated PCL were analyzed. Irradiation causes an increase in the compression modulus of PCL. This process occurs at the pre-gelation stage and continues in the gel-containing system. We have demonstrated, for the first time, that irradiation of solid PCL is accompanied by a pronounced post-effect, which manifests itself by changes in the average molecular weight. EPR data indicate that this effect, at least in part, is caused by the presence of long-lived radicals trapped in the crystalline regions. Irradiation with the sterilizing dose does not cause a statistically significant change in the biocompatibility of PCL after subsequent storage for 79 d, as determined by preliminary osteoblast vitality tests.