Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

Synthesis and mechanical evaluation of Sr-doped calcium-zirconium-silicate (baghdadite) and its impact on osteoblast cell proliferation and ALP activity.

For the first time the successful preparation of Sr doped baghdadite (Ca3-x Sr x ZrSi2O9 x = 0.1 and 0.75) is shown. Sr-doped as well as pure baghdadite are prepared via a versatile solid-state synthesis and conventional sintering at 1400 °C. XRD measurements and crystal structure refinements reveal that a substitution of Ca atoms with Sr and a high purity (>99%) is achieved. The physical, mechanical, and biological properties of these novel bioceramics are presented in relation to the dopant concentration. Incorporating Sr into the baghdadite crystal caused only minor changes to the grain size and the mechanical properties. The characteristic strength ranges from 145 to 168 MPa and a Weibull modulus of 4.9 to 9.2 is observed. Other mechanical properties like fracture toughness and hardness vary from 1.23 ± 0.07 MPam(0.5) to 1.31 ± 0.12 MPam(0.5) and 7.3 ± 0.6 GPa to 8.0 ± 0.7 GPa, respectively. The in vitro cellular response of human osteoblasts showed an increase in the cell proliferation and a significantly higher alkaline phosphatase (ALP) activity with an increase in the Sr content. From the improved biological properties and the suitable mechanical performance we conclude that this material is a highly promising candidate for bone replacement material and bioactive implant coatings.

Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface.

In this study a bioinspired approach to induce self-mineralization of bone-like material on alumina surfaces is presented. The mineralizing enzyme alkaline phosphatase (ALP) is covalently immobilized by a carbodiimide-mediated chemoligation method. The enzymatic activity of immobilized ALP and its mineralization capability are investigated under acellular conditions as well as in the presence of human bone cells. Analytical, biochemical and immunohistochemical characterization show that ALP is efficiently immobilized, retains its activity and can trigger calcium phosphate mineralization on alumina at acellular conditions. In vitro cell tests demonstrate that ALP-functionalized alumina clearly boosts and enhances bone cell mineralization. Our results underpin the great potential of ALP-functionalized alumina for the development of bioactive surfaces for applications such as orthopaedic and dental implants, enabling a fast and firm implant osseointegration.