Matthias Schumacher

A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro

In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca(2+) by Sr(2+) ions during cement setting. All modified cements released 0.03-0.07 mM Sr(2+) under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca(2+) depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone.

A novel and easy-to-prepare strontium(II) modified calcium phosphate bone cement with enhanced mechanical properties

The aim of this study was to evaluate two different approaches to obtaining strontium-modified calcium phosphate bone cements (SrCPCs) without elaborate synthesis of Sr-containing calcium phosphate species as cement precursors that could release biologically effective doses of Sr(2+) and thus could improve the healing of osteoporotic bone defects. Using strontium carbonate as a strontium(II) source, it was introduced into a hydroxyapatite-forming cement either by the addition of SrCO3 to an α-tricalcium phosphate-based cement precursor mixture (A-type) or by substitution of CaCO3 by SrCO3 during precursor composition (S-type). The cements, obtained after setting in a water-saturated atmosphere, contained up to 2.2at.% strontium in different distribution patterns as determined by time-of-flight secondary ion mass spectrometry and energy-dispersive X-ray spectroscopy. The setting time of CPC and A-type cements was in the range of 6.5-7.5min and increased for substitution-type cements (12.5-13.0min). Set cements had an open porosity between 26 and 42%. Compressive strength was found to increase from 29MPa up to 90% in substituted S-type cements (58MPa). SrCPC samples released between 0.45 and 1.53mgg(-1) Sr(2+) within 21days and showed increased radiopacity. Based on these findings, the SrCPC developed in this study could be beneficial for the treatment of defects of systemically impaired (e.g. osteoporotic) bone.

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.

Strontium modified calcium phosphate cements – approaches towards targeted stimulation of bone turnover

Making use of the potential of calcium phosphates to host a variety of ions in their crystal lattice, ion substitution of calcium phosphate bone cements has become the subject of intense investigations in the last few years, since this approach allows one to stabilize a bone defect and to locally deliver therapeutic ions into a specific defect site at the same time. In this respect significant attention has been given to strontium ions (Sr2+) lately. Strontium possesses the unique potential to both stimulate new bone formation and inhibit cell-driven bone resorption and thus has been used successfully in systemic osteoporosis therapy. Strontium doping of calcium phosphate bone cements might allow making use of this dual effect to promote local bone defect healing. The goal of this review is to provide an overview of different routes that have been employed to obtain strontium-containing calcium phosphate bone cements and describe their material characteristics as well as their biological properties based on cell culture and animal studies.

Strontium substitution in apatitic CaP cements effectively attenuates osteoclastic resorption but does not inhibit osteoclastogenesis.

UNLABELLED Strontium ions were discovered to exert a dual effect on bone turnover, namely an inhibition of cell-driven bone resorption and a simultaneous stimulation of new bone tissue formation. A variety of strontium containing calcium phosphate bone cements (SrCPC) have been developed to benefit from both effects to locally support the healing of osteoporotic bone defects. While the stimulating effect of strontium modification on bone forming cells has been demonstrated in a number of studies, this study focuses on the inhibition and/or reduction of osteoclastogenesis and osteoclastic resorption by a strontium substituted calcium phosphate bone cement (SrCPC). Human peripheral blood mononuclear cells (PBMC) were differentiated into osteoclasts in the presence of different Sr(2+)-concentrations as well as on the surface of SrCPC disks. Osteoclastogenesis of PBMC was shown to be merely unaffected by medium Sr(2+)-concentrations comparable to those released from SrCPC in vitro (0.05-0.15mM). However, an altering effect of 0.1mM strontium on the cytoskeleton of osteoclast-like cells was shown. In direct contact to SrCPC disks, these cells exhibited typical morphological features and osteoclast markers on both RNA and protein level were formed. However, calcium phosphate resorption was significantly decreased on strontium-containing cements in comparison to a strontium-free control. This was accompanied by an intracellular accumulation of strontium that increased with substrate strontium content as demonstrated by Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS). This study illustrates that SrCPC do not inhibit osteoclastogenesis but significantly attenuate osteoclastic substrate resorption in vitro. STATEMENT OF SIGNIFICANCE Strontium ions have been shown to promote bone formation and inhibit bone resorption. Therefore strontium is successfully used in the treatment of osteoporosis and also inspired the development of strontium-containing strontium/calcium phosphate bone cements (SrCPC). Studies have shown the positive effects of SrCPC on bone formation, however, the inhibiting effect of strontium on bone resorption in the context of such cements has not been shown so far. We found that the formation of bone-resorbing osteoclasts is not inhibited, but that their resorption activity is decreased in contact to SrCPC. The former is important since those cells play an important role in the bone cell signaling. The latter is a key requirement in osteoporosis therapy, which addresses excess bone resorption.

Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature.

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO2 (scCO2) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO2 treatment effectively inactivates microorganisms including bacterial spores. We established a scCO2 sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO2 sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO2 treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO2 treatment of these materials and scaffolds. We conclude that scCO2 sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.

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.

Dendritic Glycopolymer as Drug Delivery System for Proteasome Inhibitor Bortezomib in a Calcium Phosphate Bone Cement: First Steps Toward a Local Therapy of Osteolytic Bone Lesions

Establishment of drug delivery system (DDS) in bone substitute materials for local treatment of bone defects still requires ambitious solutions for a retarded drug release. We present two novel DDS, a weakly cationic dendritic glycopolymer and a cationic polyelectrolyte complex, composed of dendritic glycopolymer and cellulose sulfate, for the proteasome inhibitor bortezomib. Both DDS are able to induce short-term retarded release of bortezomib from calcium phosphate bone cement in comparison to a burst-release of the drug from bone cement alone. Different release parameters have been evaluated to get a first insight into the release mechanism from bone cements. In addition, biocompatibility of the calcium phosphate cement, modified with the new DDS was investigated using human mesenchymal stromal cells.

Preliminary evaluation of different biomaterials for defect healing in an experimental osteoporotic rat model with dynamic PET-CT (dPET-CT) using F-18-Sodium Fluoride (NaF)

UNLABELLED The aim of the current study was to measure and compare the effect of calcium phosphate cement (CPC) and CPC enriched with strontium (SrCPC) for the healing of osteoporotic bone defects in the rat femur using (18)F-Sodium Fluoride dPET-CT. METHODS Osteoporosis was induced by ovariectomy and a calcium restricted diet. After three months, rats were operated to create a 4 mm defect in the distal metaphyseal femur with internal fixation. 7 Rats have been treated either with CPC (Group 2) or with SrCPC (Group 3) for bone replacement and defect healing. Furthermore, a control group of 7 rats without any biomaterial (Group 1) was used for reference. 18 weeks after osteoporosis induction and 6 weeks following femoral surgery, dPET-CT studies scan were performed with (18)F-Sodium Fluoride. SUVs and a 2-tissue compartmental learning-machine model (K1-k4, VB, influx) were used for quantitative analysis. RESULTS VB, reflecting the fractional blood volume and k3, reflecting the formation of fluoroapatite were the most sensitive parameters for the characterisation of healing process and revealed the best differentiation for the control group and the CPC group (Group 2) as well as for the CPC with strontium carbonate group (Group 3) (p<0.05). VB was decreased by the order of Group 1, Group 2 and Group 3, while k3 was increased by the same order. Therefore, the data direct to a decreased fractional blood volume and increased fixation of fluoride in rats with these biomaterials. CONCLUSION We found PET scanning using (18)F-Sodium Fluoride to be a sensitive and useful method for evaluation of bone healing after replacement with CPC or SrCPC.

Formation of osteoclasts on calcium phosphate bone cements and polystyrene depends on monocyte isolation conditions.

OBJECTIVES Peripheral blood mononuclear cells (PBMC) are an attractive source for the generation of osteoclasts in vitro, which is an important prerequisite for the examination of resorption and remodeling of biomaterials. In this study, different preparation methods are used to obtain cell populations with a rising content of CD14(+) monocytes. We wanted to address the question whether there is a correlation between content of CD14(+) cells in the preparation and functionality of formed osteoclasts. MATERIALS AND METHODS PBMC obtained by density gradient centrifugation with and without further purification by plastic adherence or immunomagnetic separation of CD14(+) cells were seeded on both cell culture polystyrene and a calcium phosphate bone cement (CPC) and cultivated under stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Cell cultures were characterized by histological and fluorescent staining of multinucleated cells that were positive for tartrate-resistant acid phosphatase (TRAP) activity and the presence of actin rings, respectively. Furthermore, activities of osteoclast marker enzymes TRAP and carbonic anhydrase II (CA II) were quantified. For osteoclasts cultured on CPC, resorption pits were visualized using scanning electron microscopy (SEM). RESULTS Monocytes of all preparations were successfully differentiated into multinucleated osteoclasts showing TRAP and CA II activity on both cell culture plastic and CPC. Preparations involving an additional plastic adherence step exhibited only a minor increase of TRAP and CA II activity in the second week of cultivation. Furthermore, the number of resorption pits on CPC was reduced in these cultures compared with immunomagnetically enriched monocytes and preparations without additional plastic adherence steps. Optimal results with regard to yield, number of multinucleated osteoclasts, activity of TRAP and CA II, and resorption of CPC were obtained by simple density gradient centrifugation. CONCLUSION All examined monocyte preparation protocols were suitable for the generation of osteoclasts on both polystyrene and CPC. Highly purified monocytes are not mandatory to obtain functional osteoclasts for investigation of biomaterial resorption.