Modified Titanium Surface-Mediated Effects on Human Bone Marrow Stromal Cell Response

Surface modification of titanium implants is used to enhance osseointegration. The study objective was to evaluate five modified titanium surfaces in terms of cytocompatibility and pro-osteogenic/pro-angiogenic properties for human mesenchymal stromal cells: amorphous microporous silica (AMS), bone morphogenetic protein-2 immobilized on AMS (AMS + BMP), bio-active glass (BAG) and two titanium coatings with different porosity (T1; T2). Four surfaces served as controls: uncoated Ti (Ti), Ti functionalized with BMP-2 (Ti + BMP), Ti surface with a thickened titanium oxide layer (TiO2) and a tissue culture polystyrene surface (TCPS). The proliferation of eGFP-fLuc (enhanced green fluorescence protein-firefly luciferase) transfected cells was tracked non-invasively by fluorescence microscopy and bio-luminescence imaging. The implant surface-mediated effects on cell differentiation potential was tracked by determination of osteogenic and angiogenic parameters [alkaline phosphatase (ALP); osteocalcin (OC); osteoprotegerin (OPG); vascular endothelial growth factor-A (VEGF-A)]. Unrestrained cell proliferation was observed on (un)functionalized Ti and AMS surfaces, whereas BAG and porous titanium coatings T1 and T2 did not support cell proliferation. An important pro-osteogenic and pro-angiogenic potential of the AMS + BMP surface was observed. In contrast, coating the Ti surface with BMP did not affect the osteogenic differentiation of the progenitor cells. A significantly slower BMP-2 release from AMS compared to Ti supports these findings. In the unfunctionalized state, Ti was found to be superior to AMS in terms of OPG and VEGF-A production. AMS is suggested to be a promising implant coating material for bioactive agents delivery.


hMSC Viral Transfection
hMSCs were genetically modified in one single gene transfer step using a double reporter, i.e., eGFP (enhanced Green Fluorescent Protein) and fLuc (firefly Luciferase) lentiviral vector [3]. hMSCs were plated at density of 8 × 10 3 cells/cm 2 and cultured under standard cell culture conditions for 24 h. The cells were then rinsed twice with phosphate buffered saline (PBS), infected with 2 × 10 6 viral particles/cm 2 (250 particles/cell) added to complete αMEM medium and cultured again under standard conditions for 24 h. After 2 medium changes over 48 h, cells were observed by fluorescence microscope and further expanded. At confluence, cells were detached by trypsinization and characterized.

hMSC Characterization by Flow Cytometry
1.3.1. Phenotypic Analysis hMSCs were phenotypically characterized by fluorescence-activated cell sorting (FACS) according to stipulated criterion for hMSCs [4]. FACS analysis (Guava easyCyte, Millipore, Molsheim, France) was performed using Phycoerythrin (PE)-conjugated CD45, CD73 and CD105 antibodies (Immunotools, Friesoythe, Germany) to confirm that the phenotype of both non-transfected and transfected hMSCs was maintained after expansion in the culture. PE-conjugated isotype matched negative control antibodies were added. Fixed hMSCs (prepared from trypsinized monolayers at passage 6) were incubated with antibodies against each surface marker for 45 min at 4 °C. Cells were resuspended in PBS with 1% bovine serum albumin (BSA) and analyzed using Cytosoft software (Millipore).

Proliferation Potential Analysis
The proliferation of non-transfected versus transfected hMSCs was evaluated by seeding the cells (5 × 10 3 cells/cm 2 ) in 24 well plates (n = 3) in basic cell culture medium (αMEM with 10% FBS, 1% antibiotic and anti-mycotic and L-Glutamine at 0.292 g/L; all from Sigma, Bornem, Belgium). Cell numbers were determined after 1, 3 and 6 days of cell seeding by FACS on fixated samples.

Bioluminescence Imaging for Monitoring Cell Proliferation
For the purpose of cell proliferation evaluation by bioluminescence imaging (BLI) [5], determination of the correlation between the cell number and the bioluminescent intensity signal was performed prior to experimentation with modified titanium surfaces. Cell suspensions at serial dilutions were added to a black 96-wells plate at densities between 0 and 5 × 10 5 cells/well. D-luciferin solution (Promega, Leiden, The Netherlands) at a concentration of 200 µg/mL was added and bioluminescence images were acquired by IVIS100 optical imaging system (Xenogen Corporation, Alameda, CA, USA) with 2 s exposure time and small binning setting. BLI (expressed in # photons/s for a defined area) was determined with the help of LivingImage ® software (version 2.50.1, Xenogen Corporation). The experiment was carried out in duplicate. Background signal was subtracted from measured BLI signal and obtained value was plotted against the number of cells. Figure S1 illustrates phenotypic characterization results for non-transfected and eGFP-fLuc-transfected hMSCs. FACS analysis showed that the cells were negative for CD45 expression and positive for CD73 and CD105, a phenotype characteristic for hMSCs. Viral cell transfection did not alter the expression of these surface markers. Figure S1. Flow cytometric analyses for CD45, CD73 and CD105 surface marker expressions for (a) non-transfected (NT-hMSC) and (b) transfected (eGFP-fLuc-hMSC). Gray line indicates the control of CD marker isotypes.

Efficacy of Viral Transfection-Proliferation Potential
An efficacy of viral transfection of up to 60% was recorded, as determined by FACS. The transfection ratio remained constant during cell proliferation (as displayed in Figure S2a) as well as over different passages (results not shown). Furthermore, no differences could be observed in cell proliferation rate of both cell groups ( Figure S2b). Human MSCs were selected for use in the in vitro cell culture experiments. In order to prove the cells' nature, surface marker expression verification for hMSCs [4] was carried out. Furthermore, it was shown that lentiviral transfection did not alter phenotypic expressions of isolated cells, neither their proliferation behavior. Therefore, eGFP-fLuc transfected human bone marrow derived stromal cells were considered appropriate for evaluation of the biocompatibility of specific modified Ti surfaces and for exploration of their pro-osteogenic properties.

Bioluminescence Imaging for Monitoring Cell Proliferation
In order to use BLI as a tool to monitor cell proliferation, a correlation between BLI signal and actual cell number was checked. The correlation between emitted signal and cell number was found to be linear (R 2 = 0.996 and R 2 = 0.995 for duplicate experiments) ( Figure S3). The linear correlation between cell number and BLI signal indicate that BLI method was suitable for monitoring cell proliferation on modified Ti surfaces.