Berthold Nies

Bioactive silica-collagen composite xerogels modified by calcium phosphate phases with adjustable mechanical properties for bone replacement.

The development of composites has been recognized as a promising strategy to fulfil the complex requirements of biomaterials. The present study reports on the modification of a novel silica-collagen composite material by varying the inorganic/organic mass ratio and introducing calcium phosphate cement (CPC) as a third component. The sol-gel technique is used for processing, followed by xerogel formation under specific temperature and relative humidity conditions. Cylindrical monolithic samples up to 400mm(3) were obtained without any sintering processes. Various hierarchical phases of the organic component were applied, ranging from tropocollagen and collagen fibrils up to collagen fibers, each characterized by atomic force microscopy. Focusing on the application of fibrils, various inorganic/organic mass ratios were used: 100/0, 85/15 and 70/30; their influence on the structure of the composite material was demonstrated by scanning electron microscopy. The composition was extended by the addition of 25wt.% CPC which led to increased bioactivity by accelerating the formation of bone apatite layers in simulated body fluid. Synchrotron microcomputed tomography demonstrated the homogeneous distribution of the cement particles in the silica-collagen matrix. Compressive strength tests showed that the mechanical properties of the brittle pure silica gel are changed significantly due to collagen addition. The highest ultimate strength of about 115MPa at about 18% total strain was registered for the 70/30 silica-collagen composite xerogels. Incorporation of CPC lowered the gels strength. By demonstrating differentiation of human monocytes into osteoclast-like cells, an important feature of the composite material regarding successful bone remodeling is fulfilled.

Fabrication of porous scaffolds by three‐dimensional plotting of a pasty calcium phosphate bone cement under mild conditions

The major advantage of hydroxyapatite (HA)‐forming calcium phosphate cements (CPCs) used as bone replacement materials is their setting under physiological conditions without the necessity for thermal treatment that allows the incorporation of biological factors. In the present study, we have combined the biocompatible consolidation of CPCs with the potential of rapid prototyping (RP) techniques to generate calcium phosphate‐based scaffolds with defined inner and outer morphology. We demonstrate the application of the RP technique three‐dimensional (3D) plotting for the fabrication of HA cement scaffolds. This was realized by utilizing a paste‐like CPC (P‐CPC) which is stable as a malleable paste and whose setting reaction is initiated only after contact with aqueous solutions. The P‐CPC showed good processability in the 3D plotting process and allowed the fabrication of stable 3D structures of different geometries with adequate mechanical stability and compressive strength. The cytocompatibility of the plotted P‐CPC scaffolds was demonstrated in a cell culture experiment with human mesenchymal stem cells. The mild conditions during 3D plotting and post‐processing and the realization of the whole procedure under sterile conditions make this approach highly attractive for fabrication of individualized implants with respect to patient‐specific requirements by simultaneous plotting of biological components. Copyright

Ready-to-use injectable calcium phosphate bone cement paste as drug carrier

Current developments in calcium phosphate cement (CPC) technology concern the use of ready-to-use injectable cement pastes by dispersing the cement powder in a water-miscible solvent, such that, after injection into the physiological environment, setting of cements occurs by diffusion of water into the cement paste. It has also been demonstrated recently that the combination of a water-immiscible carrier liquid combined with suitable surfactants facilitates a discontinuous liquid exchange in CPC, enabling the cement setting reaction to take place. This paper reports on the use of these novel cement paste formulations as a controlled release system of antibiotics (gentamicin, vancomycin). Cement pastes were applied either as a one-component material, in which the solid drugs were physically dispersed, or as a two-component system, where the drugs were dissolved in an aqueous phase that was homogeneously mixed with the cement paste using a static mixing device during injection. Drug release profiles of both antibiotics from pre-mixed one- and two-component cements were characterized by an initial burst release of ∼7-28%, followed by a typical square root of time release kinetic for vancomycin. Gentamicin release rates also decreased during the first days of the release study, but after ∼1 week, the release rates were more or less constant over a period of several weeks. This anomalous release kinetic was attributed to participation of the sulfate counter ion in the cement setting reaction altering the drug solubility. The drug-loaded cement pastes showed high antimicrobial potency against Staphylococcus aureus in an agar diffusion test regime, while other cement properties such as mechanical performance or phase composition after setting were only marginally affected.

Well-ordered biphasic calcium phosphate–alginate scaffolds fabricated by multi-channel 3D plotting under mild conditions

Herein, we present a new type of biphasic organic-inorganic scaffold, which can be fabricated by multi-channel 3D plotting under mild conditions based on a highly concentrated alginate paste and a ready-to-use calcium phosphate cement (CPC) for bone and osteochondral tissue engineering. The structures of scaffolds were characterised by light and scanning electron microscopy (SEM). Results indicated that the concentrated alginate and CPC pastes had comparable plotting consistency, and therefore could be combined in one (biphasic) scaffold by applying predesigned plotting parameters. After crosslinking of alginate and setting of CPC, the biphasic scaffold obtained mechanical and structural stability. Mechanical test data revealed that biphasic CPC-alginate scaffolds had significantly increased compressive strength and modulus compared to pure alginate as well as mixed calcium phosphate (CaP)-alginate scaffolds in a wet state and improved strength and toughness compared to pure CPC scaffolds in both dry and wet conditions. Culture of human mesenchymal stem cells (hMSCs) on these scaffolds over 3 weeks demonstrated the good cytocompatibility of the selected materials. Because of the mild preparation conditions, bovine serum albumin (BSA) as a model protein was loaded in alginate and CPC pastes prior to plotting with high loading efficiency. Release studies in vitro showed that BSA released much faster from alginate strands than from CPC strands, which might allow amount-controlled protein release from biphasic CPC-alginate scaffolds. Furthermore, an upgraded bipartite osteochondral scaffold consisting of an alginate part for chondral and a biphasic CPC-alginate part for bony repair was fabricated based on this technique. This scaffold showed a strong organic-inorganic interface binding due to interlocking and crosslinking of the alginate strands.

3D plotting of growth factor loaded calcium phosphate cement scaffolds.

UNLABELLED Additive manufacturing allows to widely control the geometrical features of implants. Recently, we described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of a storable CPC paste based on water-immiscible carrier liquid. Plotting and hardening is conducted under mild conditions allowing the (precise and local) integration of biological components. In this study, we have developed a procedure for efficient loading of growth factors in the CPC scaffolds during plotting and demonstrated the feasibility of this approach. Bovine serum albumin (BSA) or vascular endothelial growth factor (VEGF), used as model proteins, were encapsulated in chitosan/dextran sulphate microparticles which could be easily mixed into the CPC paste in freeze-dried state. In order to prevent leaching of the proteins during cement setting, usually carried out by immersion in aqueous solutions, the plotted scaffolds were aged in water-saturated atmosphere (humidity). Setting in humidity avoided early loss of loaded proteins but provided sufficient amount of water to allow cement setting, as indicated by XRD analysis and mechanical testing in comparison to scaffolds set in water. Moreover, humidity-set scaffolds were characterised by altered, even improved properties: no swelling or crack formation was observed and accordingly, surface topography, total porosity and compressive modulus of the humidity-set scaffolds differed from those of the water-set counterparts. Direct cultivation of mesenchymal stem cells on the humidity-set scaffolds over 21days revealed their cytocompatibility. Maintenance of the bioactivity of VEGF during the fabrication procedure was proven in indirect and direct culture experiments with endothelial cells. STATEMENT OF SIGNIFICANCE Additive manufacturing techniques allow the fabrication of implants with defined architecture (inner pore structure and outer shape). Especially printing technologies conducted under mild conditions allow additionally the (spatially controlled) integration of biological components such as drugs or growth factors. That enables the generation of individualized implants which can better meet the requirements of a patient and of tissue engineering constructs. To our knowledge, simultaneous printing of biological components was up to now only described for hydrogel/biopolymer-based materials which suffer from poor mechanical properties. In contrast, we have developed a procedure (based on 3D plotting of a calcium phosphate cement paste) for the fabrication of designed and growth factor loaded calcium-phosphate-based scaffolds applicable for bone regeneration.

Properties of injectable ready-to-use calcium phosphate cement based on water-immiscible liquid.

Calcium phosphate cements (CPCs) are highly valuable materials for filling bone defects and bone augmentation by minimal invasive application via percutaneous injection. In the present study some key features were significantly improved by developing a novel injectable ready-to-use calcium phosphate cement based on water-immiscible carrier liquids. A combination of two surfactants was identified to facilitate the targeted discontinuous exchange of the liquid for water after contact with aqueous solutions, enabling the setting reaction to take place at distinct ratios of cement components to water. This prolonged the shelf life of the pre-mixed paste and enhanced reproducibility during application and setting reactions. The developed paste technology is applicable for different CPC formulations. Evaluations were performed for the formulation of an α-TCP-based CPC as a representative example for the preparation of injectable pastes with a powder-to-carrier liquid ratio of up to 85:15. We demonstrate that the resulting material retains the desirable properties of conventional CPC counterparts for fast setting, mechanical strength and biocompatibility, shows improved cohesion and will most probably show a similar degree of resorbability due to identical mineral structure of the set products.

Physicochemical and cell biological characterization of PMMA bone cements modified with additives to increase bioactivity

Polymethylmethacrylate (PMMA) bone cement is the most widely used material in surgery to fix joint replacements in the bone. In this study, we propose a new approach to generate bioactive PMMA surfaces directly at the site of implantation by adding the amphiphilic molecule phosphorylated 2-hydroxyethylmethacrylate (HEMA-P) to commercial PMMA bone cement, both with or without addition of 1-5% soluble calcium and carbonate salts. The setting behavior as well as the mechanical properties, the bonding quality at the metal/cement interface, mineral deposition, and cellular response for different cement modifications were investigated in vitro. The addition of HEMA-P resulted in entirely positive effects with respect to proliferation and differentiation of osteoblast-like cells (SaOs-2) and a very tight contact at the metal/cement interface. No detrimental changes of other properties were detected. The additional incorporation of salts provoked an increased deposition of calcium phosphate minerals but no further improvement in SaOs-2 cell differentiation. A significant decrease in polarization resistance for cements with high salt content (5%) was attributed to debonding between metal and cement. The results suggest an improved clinical performance of PMMA/HEMA-P composites, which might be further enhanced by small amounts of the soluble salts.

Mechanical Properties and Drug Release Behavior of Bioactivated PMMA Cements

Septic loosening of cemented implants represents an unresolved long-term problem of total hip endoprostheses. Common treatments of infected prostheses involve the use of temporary antibiotic-loaded PMMA spacer-implants or antibiotic-loaded cements. The latter are either provided by a manufacturer or are obtained by simply mixing specific antibiotic powders according to a microbial sensitivity test with PMMA cement. This study is aimed to investigate the antibiotic release behavior and mechanical properties of novel modified PMMA cements, which were bioactivated by chemical modification of commercial cements with either 0.5% hydroxyethylmethacrylate-phosphate (HEMA-P) or 0.5% hydroxyethylmethacrylate-phosphate + calcium chloride and sodium carbonate as buffer. Tobramycin release experiments from the cements were performed statically by immersion of the drug-loaded samples in PBS buffer following liquid change after different periods of time or during cyclic mechanical loading of the cement samples. Cement modification did not significantly alter the mechanical properties of the cements, but affected the release rate from the matrix. While the unmodified cement released approximately 0.33 mg/cm2 tobramycin after 48 h independent of the testing regime, modification with both HEMA-P and salt buffer increased the antibiotic release to 37–50 mg/cm2 when tested under cyclical mechanical loading.

Modifications of a calcium phosphate cement with biomolecules--influence on nanostructure, material, and biological properties.

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding.

Formation of nano hydroxyapatite - : a straightforward way to bioactivate bone implant surfaces

Abstract Bioactivity has been a field of biomaterials research for more than 25 years. However, clinical application of bioactive implants is progressing rather slowly and is still limited to a few types of bone implants. The increased understanding of the molecular mechanisms underlying bioactivity has opened up new attractive opportunities to render conventional implant materials such as metals, ceramics and even polymers bioactive. In particular, biomimetic nano hydroxyapatite coatings and in-situ mineralising surfaces induced by incorporation of mineralisation seeds are applicable to a wide variety of implant types and materials. They have the potential to set new standards for the improved performance of orthopaedic implants whilst complying with the economic constraints on healthcare.