Ulrike Deisinger

In vitro -Osteoclastic Activity Studies on Surfaces of 3D Printed Calcium Phosphate Scaffolds:

Various biomaterials have been developed for the use as bone substitutes for bone defects. To optimize their integration and functionality, they should be adapted to the individual defect. Rapid prototyping is a manufacturing method to tailor materials to the 3D geometry of the defect. Especially 3D printing allows the manufacture of implants, the shape of which can be designed to fit the bone defect using anatomical information obtained from the patient. 3D printing of calcium phosphates, which are well established as bone substitutes, involves a sintering step after gluing the granules together by a binder liquid. In this study, we analyzed if and how these 3D printed calcium phosphate surfaces can be resorbed by osteoclast-like cells. On 3D printed scaffold surfaces consisting of pure HA and β-TCP as well as a biphasic mixture of HA and TCP the osteoclastic cell differentiation was studied. In this regard, cell proliferation, differentiation, and activation were analyzed with the monocytic cell line RAW 264.7. The results show that osteoclast-like cells were able to resorb calcium phosphate surfaces consisting of granules. Furthermore, biphasic calcium phosphate ceramics exhibit, because of their osteoclastic activation ability, the most promising surface properties to serve as 3D printed bone substitute scaffolds.

The chemical composition of synthetic bone substitutes influences tissue reactions in vivo: histological and histomorphometrical analysis of the cellular inflammatory response to hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate ceramics

Bone substitute material properties such as granule size, macroporosity, microporosity and shape have been shown to influence the cellular inflammatory response to a bone substitute material. Keeping these parameters constant, the present study analyzed the in vivo tissue reaction to three bone substitute materials (granules) with different chemical compositions (hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and a mixture of both with a HA/TCP ratio of 60/40 wt%). Using a subcutaneous implantation model in Wistar rats for up to 30 days, tissue reactions, including the induction of multinucleated giant cells and the extent of implantation bed vascularization, were assessed using histological and histomorphometrical analyses. The results showed that the chemical composition of the bone substitute material significantly influenced the cellular response. When compared to HA, TCP attracted significantly greater multinucleated giant cell formations within the implantation bed. Furthermore, the vascularization of the implantation bed of TCP was significantly higher than that of HA implantation beds. The biphasic bone substitute group combined the properties of both groups. Within the first 15 days, high giant cell formation and vascularization rates were observed, which were comparable to the TCP-group. However, after 15 days, the tissue reaction, i.e. the extent of multinucleated giant cell formation and vascularization, was comparable to the HA-group. In conclusion, the combination of both compounds HA and TCP may be a useful combination for generating a scaffold for rapid vascularization and integration during the early time points after implantation and for setting up a relatively slow degradation. Both of these factors are necessary for successful bone tissue regeneration.

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.

Fabrication of Tailored Hydroxyapatite Scaffolds: Comparison between a Direct and an Indirect Rapid Prototyping Technique

In the last few years new fabrication methods, called rapid prototyping (RP) techniques, have been developed for the fabrication of hydroxyapatite scaffolds for bone substitutes or tissue engineering applications. With this generative fabrication technology an individual tailoring of the scaffold characteristics can be realised. In this work two RP techniques, a direct (dispense-plotting) and an indirect one (negative mould technique), are described by means of fabricating hydroxyapatite (HA) scaffolds for bone substitutes or bone tissue engineering. The produced scaffolds were characterised, mainly regarding their pore and strut characteristics. By these data the performance of the two fabrication techniques was compared. Dispense-plotting turned out to be the faster technique while the negative mould method was better suited for the fabrication of exact pore and strut geometries.

Generating Porous Ceramic Scaffolds: Processing and Properties

For tissue regeneration in medicine three-dimensional scaffolds with specific characteristics are required. A very important property is a high, interconnecting porosity to enable tissue ingrowth into the scaffold. Pore size distribution and pore geometry should be adapted to the respective tissue. Additionally, the scaffolds should have a basic stability for handling during implantation, which is provided by ceramic scaffolds. Various methods to produce such ceramic 3D scaffolds exist. In this paper conventional and new fabrication techniques are reviewed. Conventional methods cover the replica of synthetic and natural templates, the use of sacrificial templates and direct foaming. Rapid prototyping techniques are the new methods listed in this work. They include fused deposition modelling, robocasting and dispense-plotting, ink jet printing, stereolithography, 3D-printing, selective laser sintering/melting and a negative mould technique also involving rapid prototyping. The various fabrication methods are described and the characteristics of the resulting scaffolds are pointed out. Finally, the techniques are compared to find out their disadvantages and advantages.

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(®)), β-tricalcium phosphate (β-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.

Biofunctionalization of dispense‐plotted hydroxyapatite scaffolds with peptides: Quantification and cellular response

Hydroxyapatite (HA) ceramic is a widely used synthetic bone substitute material for the regeneration of bone defects. We manufactured HA scaffolds with adjustable pore sizes and pore geometry by dispense-plotting. In addition, we attached peptides covalently onto the HA surface and are able to simultaneously quantify the amount of covalently attached and adsorbed peptide down to the picomolar range with a novel fluorescence-based detection method. In cell culture assays with stromal bone marrow cells, we observed a positive effect of biofunctionalization on cell differentiation after 21 days of culture when comparing the scaffold functionalized with the RGD motif containing adhesion peptide to an unmodified scaffold.

Development of Hydroxyapatite Ceramics with Tailored Pore Structure

The porosity of a hydroxyapatite ceramic was tailored by transfe rring polymeric pore models into ceramic forms via the slip casting technique. The rheolog ical properties of the hydroxyapatite slurries play an important role in facilitating t he casting operation. The optimised slurry with a solids content of 60 wt% hydroxyapatite had a slight s hear thickening flow behaviour and a low viscosity. Via impregnating of polymeric pore models and dip coating of pol ymeric foams ceramic green bodies were fabricated. After sintering at 1250 °C the polymer was burnt out and a porous ceramic was achieved. This porous ceramic could be reasonably handl ed and had a very high interconnecting porosity of 91 – 96 vol%. The pore size varied between 300 and 800 μm. The density value of the bulk hydroxyapatite ceramic covered the range bet w en 94 and 96 % th.d.. The characteristics of the porous ceramics could be varied between high and undirected porosity on the one hand, and lower porosity with defined pore channels in the three dimensiona l directions on the other hand. Introduction There is an increasing clinical requirement for bone graft mate rial, because there are many possible applications such as revision hip surgery, defect filling after e.g . a tumor surgery, or reconstructive orthopaedic surgery. As hydroxyapatite closely resembles the miner al phase of natural bone and is highly biocompatible, it is among other calcium phosphates widely used a s bone substitute material [1]. Synthetic hydroxyapatite can be reproducibly processed, and additionally, pr ovides no danger of infections. A large number of research activities has been dealing with the fabrication of synthetic hydroxyapatite ceramics [2, 3, 4, 5]. In this work the porosity of a hydroxyapatite ceramic was tailore d, so that the resulting bone substitute material is adapted to the requirements of the implanta tion site. The tailoring of the pore structure was carried out by using commercially available polyme ric foams and polymeric pore models, which were produced via rapid prototyping. These polymeric models w ere coated or impregnated with hydroxyapatite using the slip casting technique. This work is part of the research network ForTePro (Bayerische F orschungsstiftung, Germany), in which scientists of different research areas, particularly medical doctors, develop individually adapted implants for bone and cartilage defects, which will be cultivated with the pat ients own cells. Materials and Methods For producing the slurry the commercially available hydroxyapatite powder from Merck, Germany, was used. The specific surface area was characterised by the BET method (model Gemini 2370, micromeritics, Germany). The mean particle size d 50 was determined with a laser particle size analyser (model Granulomètre 850, Cilas Alcatel, France). The powder as slowly added to distilled water under constant stirring. The dispersant agent was a solution of a natrium salt of an acrylic acid copolymer. Additionally, a surfactant for improved wett ability and a binder were used. Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 977-980 doi:10.4028/www.scientific.net/KEM.254-256.977

In Vitro Studies of Cell Growth on Three Differently Fabricated Hydroxyapatite Ceramic Scaffolds for Bone Tissue Engineering

The aim of this study is to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. Therefore, three methods were used (a polyurethane (PU)-replica technique, the dispense-plotting and a negative mould technique) to produce porous hydroxyapatite (HA) ceramics. The different HA-scaffolds were then cultivated with an osteoblastic precursor cell line. In our study, highest cell proliferation and differentiation was achieved by using (PU)-replica technique. However, this study shows also that all three types of scaffolds are suitable for tissue engineering applications and as bone substitute material. The knowledge about the influence of pore size and geometry on the cell behaviour will help to tailor scaffolds, by different 3D fabrication methods, for the needs of tissue engineering laboratories or patients.

Hydroxyapatite Ceramics with Tailored Pore Structure

The porosity of a hydroxyapatite ceramic was tailored by transferring polymeric pore models into ceramic forms via the slip casting technique. The rheological properties of the hydroxyapatite slurries play an important role in facilitating the casting operation. Aqueous hydroxyapatite slurries with a high solids content were fabricated. The optimised slurry with 60 wt% hydroxyapatite had a slight shear thickening flow behaviour and a low viscosity. The resulting porous ceramic could be reasonably handled and had a very high interconnecting porosity of 91 – 96 vol%. The pore size varied between 300 and 800 μm. The density value of the bulk hydroxyapatite ceramic covered the range between 94 and 96 % th.d.. The characteristics of the porous ceramics could be varied between high and undirected porosity and low porosity with defined pore channels in the three dimensional directions. Introduction There is an increasing clinical requirement for bone graft material, because there are many possible applications such as revision hip surgery, defect filling after e.g. a tumor surgery, or reconstructive orthopaedic surgery. As hydroxyapatite closely resembles the mineral phase of natural bone and is highly biocompatible, it is among other calcium phosphates widely used as bone substitute material [1]. Synthetic hydroxyapatite can be reproducibly processed, and additionally, provides no danger of infections. A large number of research activities has been dealing with the fabrication of synthetic hydroxyapatite ceramics [2, 3, 4, 5]. In this work the porosity of a hydroxyapatite ceramic was tailored, so that the resulting bone substitute material is adapted to the requirements of the implantation site. The tailoring of the pore structure was carried out by using commercially available polymeric foams and polymeric pore models, which were produced via rapid prototyping. These polymeric models were coated or impregnated with hydroxyapatite using the slip casting technique. This work is part of the research network ForTePro (Bayerische Forschungsstiftung, Germany), in which scientists of different research areas, particularly medical doctors, develop individually adapted implants for bone and cartilage defects, which will be cultivated with the patient’s own cells. Materials and Methods For producing the slurry the commercially available hydroxyapatite powder from Merck, Germany, was used. The specific surface area was characterised by the BET method (model Gemini 2370, micromeritics, Germany). The mean particle size d50 was determined with a laser particle size analyser (model Granulomètre 850, Cilas Alcatel, France). The powder was slowly added to distilled water under constant stirring. The dispersant agent was a solution of a natrium salt of an acrylic acid copolymer. Additionally, a surfactant for better wettability and a binder were used. The Key Engineering Materials Online: 2004-05-15 ISSN: 1662-9795, Vols. 264-268, pp 2047-2050 doi:10.4028/www.scientific.net/KEM.264-268.2047