Potential Effects of Nonadherent on Adherent Human Umbilical Venous Endothelial Cells in Cell Culture

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.

Gelatine microbubble as bioactive porogen in calcium phosphate cement

The objective of this study was to prepare instant macroporous calcium phosphate cement (CPC) with enhanced degradation rate and improved initial cell adhesion by simply incorporating lab-made gelatine microbubble (Gel MB) as dry porogen into the cement. From the study, it was found that viscosity of the cement paste was a key parameter to produce small or large macropores in the cements. Pore size was also determined by microbubble size, which was originally controlled by gelatine concentration in a bubble fabrication process. CPC with high porosity (60%) and acceptable cement setting time could be obtained from the study by incorporating 10 wt.% gelatine into the cement. Greater number of MC3T3-cells were found on the surface of the Gel MB loaded CPCs. The increase of initial cell adhesion may be attributed to protein molecules adhered on the cement surface and increase of surface roughness after porogen disintegration. In sum, a one-step composite cement paste production, proposed in the study, may be applicable for fabricating rapid macropores in CPCs with improved cell adhesion for bone tissue engineering applications.

Biofunctionalization of Ceramic Implant Surfaces to Improve their Bone Ingrowth Behavior

Surface biofunctionalization is a common strategy to improve the material-tissue interface of inert implant surfaces. In this context we coated alumina-toughened zirconia (ATZ) ceramics after titanium plasma spraying with two different porous calcium phosphate layers and subsequently functionalized the obtained surfaces either with an RGD containing cell adhesion peptide sequence or a bone morphogenetic protein (BMP)-glycosaminoglycan complex. We studied initial cell adhesion densities, integrin expression, and alkaline phosphatase activity as an osteogenic marker of the coatings in vitro in comparison to the non-functionalized ATZ ceramics to evaluate the bone ingrowth potential of these biofunctionalized implant coatings.