Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 µm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 µm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 µm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 µm concavities, and osteocalcin staining predominantly for 440 and 800 µm concavities, but not for 1800 µm concavities and only slightly for planar surface controls.

Influence of ceramic disk material, surface hemispheres, and SBF volume on in vitro mineralization.

Calcium phosphate ceramics are the main mineral constituents of bone and teeth and have therefore been extensively investigated for bone regenerative applications. In the current study, the effect of disk material, surface geometry, and SBF volume on mineralization capacity was investigated. Hemispherical concavities were created on the surfaces of disks made of different materials (i.e., hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP), biphasic calcium phosphate (BCP) and titanium (Ti)) which were sintered at 1200 °C. Mineralization of CaP was assessed on disk surfaces after immersion of the samples in different volumes of simulated body fluid (SBF) up to 14 days by means of calcium assay and scanning electron microscopy (SEM). This study showed that different SBF volumes have different effects on mineralization, with an optimum material/liquid ratio of 5 mL of SBF per cm(2) . Additionally, at this volume, apparent differences based on disk material became obvious. Evidently, surface hemispherical concavities acted as initiator areas for nucleation and crystal growth.

Effects of calcium phosphate composition in sputter coatings on in vitro and in vivo performance.

Calcium phosphate (CaP) ceramic coatings have been used to enhance the biocompatibility and osteoconductive properties of metallic implants. The chemical composition of these ceramic coatings is an important parameter, which can influence the final bone performance of the implant. In this study, the effect of phase composition of CaP-sputtered coatings was investigated on in vitro dissolution behavior and in vivo bone response. Coatings were prepared by a radio frequency (RF) magnetron sputtering technique; three types of CaP target materials were used to obtain coatings with different stoichiometry and calcium to phosphate ratios (hydroxyapatite (HA), α-tricalciumphosphate (α-TCP), and tetracalciumphosphate (TTCP)) were compared with non-coated titanium controls. The applied ceramic coatings were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and inductively coupled plasma optical emission spectroscopy. The in vitro dissolution/precipitation of the CaP coatings was evaluated using immersion tests in simulated body fluid (SBF). To mimic the in vivo situation, identical CaP coatings were also evaluated in a femoral condyle rabbit model. TCPH and TTCPH showed morphological changes during 4-week immersion in SBF. The results of bone implant contact (BIC) and peri-implant bone volume (BV) showed a similar response for all experimental coatings. An apparent increase in tartrate resistant acid phosphatase (TRAP) positive staining was observed in the peri-implant region with decreasing coating stability. In conclusion, the experimental groups showed different coating properties when tested in vitro and an apparent increase in bone remodeling with increasing coating dissolution in vivo.

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics.

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and ß-TCP sintered disks: 1.8mm (large concavity), 0.8mm (medium concavity) and 0.4mm (small concavity). HA and ß-TCP disks were sintered at 1100 or 1200°C and soaked in simulated body fluid for 28 days at 37°C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200°C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.