Data supporting attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts

The provided data contains the phagocytic interaction of human MG-63 osteoblasts with micro-particles 6 µm in size as well as geometric micro-pillared topography with micro-pillar sizes 5 µm of length, width, height and spacing respectively related to the research article entitled “Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts” in the Biomaterials journal. [1] Micro-particle treatment was used as positive control triggering phagocytosis by the osteoblasts. Caveolin-1 (Cav-1) as major structural component of caveolae [2] plays an important role in the phagocytic process of micro-particles and -pillars. Data related to the experiments in [1] with siRNA-mediated knockdown are presented here as well as micro-particle control experiments, tubulin analysis on the micro-pillared topography and initial cell interaction with the micro-pillars.


a b s t r a c t
The provided data contains the phagocytic interaction of human MG-63 osteoblasts with micro-particles 6 mm in size as well as geometric micro-pillared topography with micro-pillar sizes 5 mm of length, width, height and spacing respectively related to the research article entitled "Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts" in the Biomaterials journal. [1] Micro-particle treatment was used as positive control triggering phagocytosis by the osteoblasts. Caveolin-1 (Cav-1) as major structural component of caveolae [2] plays an important role in the phagocytic process of microparticles and -pillars. Data related to the experiments in [1] with siRNA-mediated knockdown are presented here as well as microparticle control experiments, tubulin analysis on the micropillared topography and initial cell interaction with the micropillars.
& Visualization of protein localization changes via immuno-labeling after particle treatment and on the micro-pillared topography; cell morphology changes after micro-particles treatment Data source location University Medical Center Rostock, Germany Data accessibility Data is available in this article and related to [1] Value of the data The data inform future studies of topography-induced phagocytic responses of osteoblasts, which is of relevance for designing new implant surfaces.
Micro-pillared topography has an enormous effect on the actin arrangement but no impact on tubulin cytoskeleton organization, thus the data inform about the complexity of cellular reactions on biomaterial topographies.
Utilizing 6 mm sized particles showed triggering phagocytosis in osteoblasts with CD68 involvement and only partial Caveolin-1 dependency relevant for researches in the implant weardebris area.
The data displays the independence of Caveolin-1 on actin reorganization during phagocytosis.
1. Data, experimental design, materials and methods

Micro-particle treatment of human MG-63 cells and SEM sample preparation
MG-63 (American Type Culture Collection ATCC s , CRL-1427) were grown in Dulbecco's modified eagle medium (DMEM, Life Technologies GmbH, Darmstadt, Germany) with 10% fetal calf serum (FCS) (Biochrom FCS Superior, Merck KGaA, Darmstadt, Germany) and 1% gentamycin (Ratiopharm GmbH, Ulm, Germany) at 37°C in a humidified atmosphere with 5% CO 2 . Cells were seeded on cover glasses and incubated for 1 h at 37°C and 5% CO 2 to ensure adhesion. Afterwards the cells were incubated with melamine particles 6 mm in size marked with FITC (Sigma Aldrich) in a concentration of 10 5 ml À 1 for 24 h. For SEM sample preparations cells were washed with PBS three times and then fixed with 2.5% glutardialdehyde (Merck KGaA) for 1 h at RT, dehydrated through a graded series of ethanol (30%, 50%, 75%, 90% and 100% for 5, 5, 15, 10 min and twice for 10 min, respectively) dried in a critical point dryer (K 850, EMITECH, Taunusstein, Germany) and then samples were sputtered with gold for 100 s (layer ca. 20 nm) (SCD 004, BAL-TEC, Wetzlar, Germany).

Cav-1 transfection
Small interfering RNA (siRNA) against Cav-1 as well as control siRNA were obtained from Ambion (Life Technologies GmbH). For the transfection, 30,000 MG-63 cells were seeded in a 24-well plate and cultured overnight. Then the cells were transfected with 50 nM siRNA using MG-63 Transfection Reagent (Altogen Biosystems, Las Vegas, NV, USA) according to the manufacturer's instructions. 48 h after the transfection the cells were ready for further experiments. For once they were treated for 24 h with 6 mm particles and they were also trypsinated and seeded onto the Ti arrays for 24 h.

Micro-particle uptake and distribution
In Fig. 1 the cell morphology and actin cytoskeleton organization of human MG-63 cells after micro-particle treatment is presented. The cells phagocytize several micro-particles during 24 h incubation time. All particles were concentrated and not freely distributed inside the cells.

2.2.
Actin cytoskeleton organization after siRNA-mediated Cav-1 knockdown in MG-63 cells after microparticle treatment and on the micro-pillared topography The actin cytoskeleton after Cav-1 attenuation was arranged in short filaments around noninternalized particles, which were washed away during the preparation (Fig. 2). The images show a reduced particle phagocytosis by MG-63 cells, but no complete inhibition of the phagocytosis, as reported in the past. [4] The MG-63 osteoblasts with siRNA mediated Cav-1 knockdown grown on the micro-pillars indicated the same rearrangement of the actin cytoskeleton as seen in control cells, illustrated by Fig. 3.

CD68 localization after micro-particle phagocytosis
Immunofluorescence staining showed an enrichment of CD68 around internalized particles 6 mm in size, presented by Fig. 4. 2.4. α-Tubulin localization in MG-63 osteoblasts on micro-pillared topography

Initial cell dynamic on the micro-pillared topography
The MG-63 cells are actively testing the underlying topography with their filopodia during the first 6 h after cell seeding onto the micro-pillared topography, shown by Movie 1.