Indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: influence of phase composition, macroporosity and pore geometry on mechanical properties.

While various materials have been developed for bone substitute and bone tissue engineering applications over the last decades, processing techniques meeting the high demands of scaffold shaping are still under development. Individually adapted and mechanically optimised scaffolds can be derived from calcium phosphate (CaP-) ceramics via rapid prototyping (RP). In this study, porous ceramic scaffolds with a periodic pattern of interconnecting pores were prepared from hydroxyapatite, ß-tricalcium phosphate and biphasic calcium phosphates using a negative-mould RP technique. Moulds predetermining various pore patterns (round and square cross section, perpendicular and 60° inclined orientation) were manufactured via a wax printer and subsequently impregnated with CaP-ceramic slurries. Different pore patterns resulted in macroporosity values ranging from about 26.0-71.9 vol% with pore diameters of approximately 340 µm. Compressive strength of the specimens (1.3-27.6 MPa) was found to be mainly influenced by the phase composition as well as the macroporosity, both exceeding the influence of the pore geometry. A maximum was found for scaffolds with 60 wt% hydroxyapatite and 26.0 vol% open porosity. It has been shown that wax ink-jet printing allows to process CaP-ceramic into scaffolds with highly defined geometry, exhibiting strength values that can be adjusted by phase composition and pore geometry. This strength level is within and above the range of human cancellous bone. Therefore, this technique is well suited to manufacture scaffolds for bone tissue engineering.

Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique.

The adequate regeneration of large bone defects is still a major problem in orthopaedic surgery. Synthetic bone substitute materials have to be biocompatible, biodegradable, osteoconductive and processable into macroporous scaffolds tailored to the patient specific defect. Hydroxyapatite (HA) and tricalcium phosphate (TCP) as well as mixtures of both phases, biphasic calcium phosphate ceramics (BCP), meet all these requirements and are considered to be optimal synthetic bone substitute materials. Rapid prototyping (RP) can be applied to manufacture scaffolds, meeting the criteria required to ensure bone ingrowth such as high porosity and defined pore characteristics. Such scaffolds can be used for bone tissue engineering (BTE), a concept based on the cultivation of osteogenic cells on osteoconductive scaffolds. In this study, scaffolds with interconnecting macroporosity were manufactured from HA, TCP and BCP (60 wt% HA) using an indirect rapid prototyping technique involving wax ink-jet printing. ST-2 bone marrow stromal cells (BMSCs) were seeded onto the scaffolds and cultivated for 17 days under either static or dynamic culture conditions and osteogenic stimulation. While cell number within the scaffold pore system decreased in case of static conditions, dynamic cultivation allowed homogeneous cell growth even within deep pores of large (1,440 mm(3)) scaffolds. Osteogenic cell differentiation was most advanced on BCP scaffolds in both culture systems, while cells cultured under perfusion conditions were generally more differentiated after 17 days. Therefore, scaffolds manufactured from BCP ceramic and seeded with BMSCs using a dynamic culture system are the method of choice for bone tissue engineering.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy.

INTRODUCTION: Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. MATERIALS AND METHODS: Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), ß-tricalcium phosphate (ß-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. RESULTS: At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (p<0.003). This trend continued untill the 7th day of culture (p<0.003). The expression of vascular endothelial growth factor was enhanced in biomaterials where cell polarization predominated at the 7th day of culture (p=0.001). CONCLUSIONS: This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs.

Bone formation and degradation of a highly porous biphasic calcium phosphate ceramic in presence of BMP-7, VEGF and mesenchymal stem cells in an ectopic mouse model.

INTRODUCTION: Angiogenesis and mesenchymal stem cells (MSCs) promote osteogenesis. The aim of the present study was to evaluate whether bone morphogenetic protein (BMP-7) promoted osteoinduction could be enhanced by combining it with vascular endothelial growth factor (VEGF) or MSCs in highly porous biphasic calcium phosphate (BCP) ceramics. MATERIALS AND METHODS: BCP ceramic blocks were implanted in an ectopic site in 24 mice (BMP-7 vs. BMP-7/VEGF; BMP-7 vs. BMP-7/MSCs and BMP-7 vs. Control; each group n=8). Specimens were analysed 12 weeks after surgery by environmental scanning electron microscopy (ESEM) and Giemsa staining. RESULTS: In all implanted scaffolds, newly formed bone was observed, even in the control site. No statistical differences in the amount of new bone were found in the presence of BMP-7 compared to BMP-7/VEGF (p=1.0) or BMP-7/MSCs (p=0.786). ESEM revealed a degradation of the scaffolds. A higher degradation was observed in areas where no bone-implant contact was present compared to areas where the ceramic was integrated in newly formed bone. CONCLUSIONS: Neither VEGF nor MSCs enhanced BMP-7 induced bone formation under the selected conditions. The present ceramic seemed to be osteoinductive and degradable, making this material suitable for bone tissue engineering.

3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique.

The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.