Anja Henß

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.

Aromatic Hydroxylation in a Copper Bis(imine) Complex Mediated by a μ‐η2:η2 Peroxo Dicopper Core: A Mechanistic Scenario

Detailed mechanistic studies on the ligand hydroxylation reaction mediated by a copper bis(imine) complex are presented. Starting from a structural analysis of the CuI complex and the CuII product with a hydroxylated ligand, the optical absorption and vibrational spectra of starting material and product are analyzed. Kinetic analysis of the ligand hydroxylation reaction shows that O2 binding is the rate-limiting step. The reaction proceeds much faster in methanol than in acetonitrile. Moreover, an inverse kinetic isotope effect (KIE) is evidenced for the reaction in acetonitrile, which is attributed to a sterically congested transition state leading to the peroxo adduct. In methanol, however, no KIE is observed. A DFT analysis of the oxygenation reaction mediated by the micro-eta2:eta2 peroxo core demonstrates that the major barrier after O2 binding corresponds to electrophilic attack on the arene ring. The relevant orbital interaction occurs between the sigma* orbital of the Cu2O2 unit and the HOMO of the ligand. On the basis of the activation energy for the rate-limiting step (18.3 kcal mol(-1)) this reaction is thermally allowed, in agreement with the experimental observation. The calculations also predict the presence of a stable dienone intermediate which, however, escaped experimental detection so far. Reasons for these findings are considered. The implications of the results for the mechanism of tyrosinase are discussed.

Effects of macroporous, strontium loaded xerogel-scaffolds on new bone formation in critical-size metaphyseal fracture defects in ovariectomized rats

New bone formation was studied in a metaphyseal fracture-defect in ovariectomized rats stimulated by a plain and a strontium-enriched macroporous silica/collagen scaffold (ScB30 and ScB30Sr20) and a compact silica/collagen xerogel (B30). 45 female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) ScB30 (n=15), (2) ScB30Sr20 (n=15), and (3) B30 (n=15). 12 weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur. A 7-hole T-shaped plate at the lateral aspect of the femur stabilized the bone and the defect was filled with ScB30, ScB30Sr20 or B30 subsequently. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the ScB30Sr20 group compared to ScB30 (p=0.043) and B30 (p=0.0001) indicating an improved formation of new bone by the strontium-enriched macroporous silica/collagen scaffold. Furthermore, immunohistochemical results showed increased expression of BMP2 and OPG and a decreased RANKL expression in the ScB30Sr20 group. This was further confirmed with the gene expression analysis where an increase in prominent bone formation markers (ALP, OCN, Runx2, Col1a1 and Col10a1) was seen. No material remnants were found in the scaffold group indicating an almost complete degradation process of the biomaterials. This is confirmed by ToF-SIMS analysis that did not detect any strontium in the ScB30Sr20 group neither in the defect nor in the surrounding tissue. Taken together, this study shows the stimulating effects of strontium through increased bone formation by up regulation of osteoanabolic markers. This work also indicates the importance of material porosity, geometry and biodegradability in bone healing.

Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification.

This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.

Strontium release from Sr2+-loaded bone cements and dispersion in healthy and osteoporotic rat bone

Drug functionalization of biomaterials is a modern and popular approach in biomaterials research. Amongst others this concept is used for the functionalization of bone implants to locally stimulate the bone healing process. For example strontium ions (Sr2+) are administered in osteoporosis therapy to stimulate bone growth and have recently been integrated into bone cements. Based on results of different analytical experiments we developed a two-phase model for the transport of therapeutically active Sr2+-ions in bone in combination with Korsmeyer-Peppas kinetics for the Sr2+ release from bone cement. Data of cement dissolution experiments into water in combination with inductively coupled plasma mass spectrometry (ICP-MS) analysis account for dissolution kinetics following Noyes-Whitney rule. For dissolution in α-MEM cell culture media the process is kinetically hindered and can be described by Korsmeyer-Peppas kinetics. Time of flight secondary ion mass spectrometry (ToF-SIMS) was used to determine the Sr2+ diffusion coefficient in healthy and osteoporotic trabecular rat bone. Therefore, bone sections were dipped in aqueous Sr2+-solution by one side and the Sr2+-profile was measured by classical SIMS depth profiling. The Sr2+ mobility can be described by a simple diffusion model and we obtained diffusion coefficients of (2.28±2.97)⋅10-12cm2/s for healthy and of (1.55±0.93)⋅10-10cm2/s for osteoporotic bone. This finding can be explained by a different bone nanostructure, which was observed by focused ion beam scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM). Finally, the time and spatially resolved drug transport was calculated by finite element method for the femur of healthy and osteoporotic rats. The obtained results were compared to mass images that were obtained from sections of in vivo experiments by ToF-SIMS. The simulated data fits quite well to experimental results. The successfully applied model for the description of drug dispersion can help to reduce the number of animal experiments in the future.

Data on TOF-SIMS analysis of Cu2+, Co2+ and Cr3+ doped calcium phosphate cements

This article contains data of time of flight secondary ion mass spectrometry (TOF-SIMS) analysis of brushite-forming calcium phosphate cements doped with biologically active metal ions. This data are related to the research article “Cu2+, Co2+ and Cr3+ doping of a calcium phosphate cement influences materials properties and response of human mesenchymal stromal cells” (Schamel et al., 2017) [1]. Cu2+, Co2+ and Cr3+ doped β-tricalcium phosphate precursor powders were used to prepare cement samples. The incorporation and distribution of the metal ions in the cement matrix was visualized by imaging mass spectrometry.

Cell behavior of human mesenchymal stromal cells in response to silica/collagen based xerogels and calcium deficient culture conditions

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

Postembedding Decalcification of Mineralized Tissue Sections Preserves the Integrity of Implanted Biomaterials and Minimizes Number of Experimental Animals

Bone histology of decalcified or undecalcified samples depends on the investigation. However, in research each method provides different information to answer the scientific question. Decalcification is the first step after sample fixation and governs what analysis is later feasible on the sections. Besides, decalcification is favored for immunostaining and in situ hybridization. Otherwise, sample decalcification can be damaging to bone biomaterials implants that contains calcium or strontium. On the other hand, after decalcification mineralization cannot be assessed using histology or imaging mass spectrometry. The current study provides a solution to the hardship caused by material presence within the bone tissue. The protocol presents a possibility of gaining sequential and alternating decalcified and undecalcified sections from the same bone sample. In this manner, investigations using histology, protein signaling, in situ hybridization, and mass spectrometry on the same sample can better answer the intended research question. Indeed, decalcification of sections and grindings resulted in well-preserved sample and biomaterials integrity. Immunostaining was comparable to that of classically decalcified samples. The study offers a novel approach that incites correlative analysis on the same sample and reduces the number of processed samples whether clinical biopsies or experimental animals.

Trivalent chromium incorporated in a crystalline calcium phosphate matrix accelerates materials degradation and bone formation in vivo

Remodeling of calcium phosphate bone cements is a crucial prerequisite for their application in the treatment of large bone defects. In the present study trivalent chromium ions were incorporated into a brushite forming calcium phosphate cement in two concentrations (10 and 50 mmol/mol β-tricalcium phosphate) and implanted into a femoral defect in rats for 3 and 6 month, non-modified brushite was used as reference. Based on our previous in vitro findings indicating both an enhanced osteoclastic activity and cytocompatibility towards osteoprogenitor cells we hypothesized a higher in vivo remodeling rate of the Cr3+ doped cements compared to the reference. A significantly enhanced degradation of the modified cements was evidenced by micro computed tomography, X-ray and histological examinations. Furthermore the formation of new bone tissue after 6 month of implantation was significantly increased from 29% to 46% during remodeling of cements, doped with the higher Cr3+ amount. Time of flight secondary ion mass spectrometry (ToF-SIMS) of histological sections was applied to investigate the release of Cr3+ ions from the cement after implantation and to image their distribution in the implant region and the surrounding bone tissue. The relatively weak incorporation of chromium into the newly formed bone tissue is in agreement to the low chromium concentrations which were released from the cements in vitro. The faster degradation of the Cr3+ doped cements was also verified by ToF-SIMS. The positive effect of Cr3+ doping on both degradation and new bone formation is discussed as a synergistic effect of Cr3+ bioactivity on osteoclastic resorption on one hand and improvement of cytocompatibility and solubility by structural changes in the calcium phosphate matrix on the other hand. STATEMENT OF SIGNIFICANCE While biologically active metal ions like strontium, magnesium and zinc are increasingly applied for the modification of ceramic bone graft materials, the present study is the first report on the incorporation of low doses of trivalent chromium ions into a calcium phosphate based biomaterial and testing of its performance in bone defect regeneration in vivo. Chromium(III)-doped calcium phosphate bone cements show improved cytocompatibility and both degradation rate and new bone formation in vivo are significantly increased compared to the reference cement. This important discovery might be the starting point for the application of trivalent chromium salts for the modification of bone graft materials to increase their remodelling rate.