Alexander Hoppe

A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics.

Several inorganic materials such as special compositions of silicate glasses, glass-ceramics and calcium phosphates have been shown to be bioactive and resorbable and to exhibit appropriate mechanical properties which make them suitable for bone tissue engineering applications. However, the exact mechanism of interaction between the ionic dissolution products of such inorganic materials and human cells are not fully understood, which has prompted considerable research work in the biomaterials community during the last decade. This review comprehensively covers literature reports which have investigated specifically the effect of dissolution products of silicate bioactive glasses and glass-ceramics in relation to osteogenesis and angiogenesis. Particularly, recent advances made in fabricating dense biomaterials and scaffolds doped with trace elements (e.g. Zn, Sr, Mg, and Cu) and investigations on the effect of these elements on the scaffold biological performance are summarized and discussed in detail. Clearly, the biological response to artificial materials depends on many parameters such as chemical composition, topography, porosity and grain size. This review, however, focuses only on the ion release kinetics of the materials and the specific effect of the released ionic dissolution products on human cell behaviour, providing also a scope for future investigations and identifying specific research needs to advance the field. The biological performance of pure and doped silicate glasses, phosphate based glasses with novel specific compositions as well as several other silicate based compounds are discussed in detail. Cells investigated in the reviewed articles include human osteoblastic and osteoclastic cells as well as endothelial cells and stem cells.

A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants

The aim of this study was to propose and validate a new unified method for testing dissolution rates of bioactive glasses and their variants, and the formation of calcium phosphate layer formation on their surface, which is an indicator of bioactivity. At present, comparison in the literature is difficult as many groups use different testing protocols. An ISO standard covers the use of simulated body fluid on standard shape materials but it does not take into account that bioactive glasses can have very different specific surface areas, as for glass powders. Validation of the proposed modified test was through round robin testing and comparison to the ISO standard where appropriate. The proposed test uses fixed mass per solution volume ratio and agitated solution. The round robin study showed differences in hydroxyapatite nucleation on glasses of different composition and between glasses of the same composition but different particle size. The results were reproducible between research facilities. Researchers should use this method when testing new glasses, or their variants, to enable comparison between the literature in the future.

Therapeutic inorganic ions in bioactive glasses to enhance bone formation and beyond

Bioactive glasses (BG) are being widely used for bone tissue engineering applications due to their bioactivity (ability to form strong bonds to bone) and their stimulating effects on bone formation. Recently, progress has been made to enhance the biological impact of BGs by incorporating specific metallic ions in silicate (or phosphate) glasses, including boron, copper, cobalt, silver, zinc and strontium. This review summarizes the newest developments on novel compositions of bioactive glasses in the field of bone tissue engineering related to osteogenesis and angiogenesis. Furthermore, new applications areas for bioactive glasses, including nerve regeneration and cancer treatment, are highlighted.

Development and characterization of magnetic iron oxide nanoparticles with a cisplatin-bearing polymer coating for targeted drug delivery

A highly selective and efficient cancer therapy can be achieved using magnetically directed superparamagnetic iron oxide nanoparticles (SPIONs) bearing a sufficient amount of the therapeutic agent. In this project, SPIONs with a dextran and cisplatin-bearing hyaluronic acid coating were successfully synthesized as a novel cisplatin drug delivery system. Transmission electron microscopy images as well as X-ray diffraction analysis showed that the individual magnetite particles were around 4.5 nm in size and monocrystalline. The small crystallite sizes led to the superparamagnetic behavior of the particles, which was exemplified in their magnetization curves, acquired using superconducting quantum interference device measurements. Hyaluronic acid was bound to the initially dextran-coated SPIONs by esterification. The resulting amide bond linkage was verified using Fourier transform infrared spectroscopy. The additional polymer layer increased the vehicle size from 22 nm to 56 nm, with a hyaluronic acid to dextran to magnetite weight ratio of 51:29:20. A maximum payload of 330 μg cisplatin/mL nanoparticle suspension was achieved, thus the particle size was further increased to around 77 nm with a zeta potential of −45 mV. No signs of particle precipitation were observed over a period of at least 8 weeks. Analysis of drug-release kinetics using the dialysis tube method revealed that these were driven by inverse ligand substitution and diffusion through the polymer shell as well as enzymatic degradation of hyaluronic acid. The biological activity of the particles was investigated in a nonadherent Jurkat cell line using flow cytometry. Further, cell viability and proliferation was examined in an adherent PC-3 cell line using xCELLigence analysis. Both tests demonstrated that particles without cisplatin were biocompatible with these cells, whereas particles with the drug induced apoptosis in a dose-dependent manner, with secondary necrosis after prolonged incubation. In conclusion, combination of dextran-coated SPIONs with hyaluronic acid and cisplatin represents a promising approach for magnetic drug targeting in the treatment of cancer.

In vitro reactivity of Cu doped 45S5 Bioglass® derived scaffolds for bone tissue engineering

Cu-doped 45S5 bioactive glasses with varying Cu contents were fabricated and used to process 3D porous scaffolds via the foam replica technique. Cu-doping results in the weakening of the glass network and a decrease in its glass transition temperature. Acellular in vitro studies revealed very high bioactivity independent of Cu doping as indicated by the fast formation of a carbonated hydroxyapatite layer (CHA) on scaffold surfaces after immersion in simulated body fluid (SBF). The kinetics of the glass-ceramic scaffolds transition to an amorphous calcium phosphate layer (ACP) and the crystallisation of CHA were explored by FT-IR and SEM analyses. The elemental distribution in the scaffold/fluid interface region was monitored by the advanced micro-PIXE-RBS (particle induced X-ray emission/Rutherford backscattering spectrometry) method. Cu-containing glasses showed slower release of Si, Ca and P from the scaffold periphery, whereas traces of Cu were found incorporated in the CaP layer on the scaffold surface. Cu release kinetics from the scaffolds in SBF were found to depend on culturing conditions while highest Cu concentrations of ∼3.1 ppm and ∼4.6 ppm under static and quasi-dynamic conditions, respectively, were observed. Since Cu exhibits potential angiogenic and osteogenic properties, the Cu-containing scaffolds are suggested as promising materials for bone tissue engineering applications.

Bioactivation of biomorphous silicon carbide bone implants

Wood-derived silicon carbide (SiC) offers a specific biomorphous microstructure similar to the cellular pore microstructure of bone. Compared with bioactive ceramics such as calcium phosphate, however, silicon carbide is considered not to induce spontaneous interface bonding to living bone. Bioactivation by chemical treatment of biomorphous silicon carbide was investigated in order to accelerate osseointegration and improve bone bonding ability. Biomorphous SiC was processed from sipo (Entrandrophragma utile) wood by heating in an inert atmosphere and infiltrating the resulting carbon replica with liquid silicon melt at 1450°C. After removing excess silicon by leaching in HF/HNO₃ the biomorphous preform consisted of β-SiC with a small amount (approximately 6wt.%) of unreacted carbon. The preform was again leached in HCl/HNO₃ and finally exposed to CaCl₂ solution. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared analyses proved that oxidation of the residual carbon at the surface induced formation of carboxyl [COO⁻] groups, which triggered adsorption of Ca(2+), as confirmed by XPS and inductively coupled plasma optical emission spectroscopy measurements. A local increase in Ca(2+) concentration stimulated in vitro precipitation of Ca₅(PO₄)₃OH (HAP) on the silicon carbide preform surface during exposure to simulated body fluid, which indicates a significantly increased bone bonding activity compared with SiC.

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.

Effect of ion release from Cu-doped 45S5 Bioglass® on 3D endothelial cell morphogenesis

Both silicate-based bioactive glasses and copper ions have demonstrated angiogenic activity and therefore represent promising bioinorganic agents for the promotion of vascularization in tissue-engineered scaffolds. This study examined the effect of ionic release products from 45S5 Bioglass® doped with 0 and 2.5 wt.% CuO (BG and Cu-BG respectively) on the formation of capillary-like networks by SVEC4-10 endothelial cells (ECs) seeded in a three-dimensional (3D) type I collagen matrix. Copper and silicon release following 24h dissolution increased non-proportionally with Cu-BG concentration in cell culture medium, while calcium levels were decreased below the initial medium concentration. EC network length, connectivity, branching, quantified by means of a 3D morphometric image analysis method, as well as proliferation and metabolic activity were reduced in a dose-dependent fashion by BG and Cu-BG ionic release products. This reduction was less prominent for BG compared to an equivalent concentration of Cu-BG, which was attributed to a lower extent of silicon release and calcium consumption. Moreover, a CuCl2 dose equivalent to the highest concentration of Cu-BG exhibited no effect on ECs. In conclusion, while the previously reported pro-angiogenic activity of both Bioglass® and copper may not be reflected in a direct response of ECs, this study provides a maximum glass concentration for non-harmful angiogenic stimulation to be examined in future work.

Formation and in vitro biocompatibility of biomimetic hydroxyapatite coatings on chemically treated carbon substrates.

Carbon derived materials such as pyrolytic carbon or carbon-carbon composites (CCCs) exhibit excellent mechanical properties making them promising candidates for bone replacement. However, these materials are considered bioinert and not to induce bone formation in vivo. In this study, a two-step chemical surface treatment including etching with HCl/HNO3 solution and subsequent soaking in CaCl2 solution was applied to carbon substrates in order to activate the materials surface towards bioactive behavior. The bioactivity was proven by soaking the samples in simulated body fluid (SBF) and formation of carbonated hydroxyapatite layer (HCA), which indicates the ability of the material to bond to bone in vivo. The materials surface is shown to be functionalized through the chemical etching creating COO(-)Ca(2+) complexes on the surface as confirmed by FTIR and XPS analyses. These ionic complexes provide nucleation sites for HAp precipitation. After similar immersion time in SBF under the condition of local supersaturation the thickness and homogeneity of the HAp layer were found to depend on the chemical pretreatment with HCl/HNO3. Homogenous HAp layers with a thickness ranging from ∼ 6 to ∼ 17 μm were achieved. The proposed bioactivating treatment of carbon stimulates HAp formation in vivo and can be considered an easy biomimetic approach for coating carbon derived materials with bone-like hydroxyapatite. In vitro cell assay with osteosarcoma cells (MG-63) showed increased cell viability (+70%) on HAp coated carbon substrates as compared to uncoated reference while both materials induced ALP expression in MG-63 cells confirming the osteoblastic phenotype.

Effects of Cu-doped 45S5 bioactive glass on the lipid peroxidation-associated growth of human osteoblast-like cells in vitro.

Bioactive glass (BG) is a highly attractive material, exhibiting both osteoinductive and osteoconductive properties, which is known to provide a growth enhancing surface for bone cells. Previous studies have shown that lipid peroxidation and in particular generation of 4-hydroxynonenal (HNE) is involved in the growth of human osteoblast-like cells, HOS, on BG. Copper (Cu), which is an essential cofactor of several enzymes as well as a proangiogenic and an antimicrobial agent, is known to induce lipid peroxidation. Therefore, the enrichment of BG with Cu could potentially have beneficial effects on the growth of the bone cells. In this study, we investigated the effects of copper-doped 45S5 BG on the growth of HOS cells and the generation of HNE. Our results confirmed the association of HNE with the growth of HOS cells. The effects of added Cu were dose-dependent. Specifically, low concentrations (i.e., 0.1% w/w) of Cu improved viability and enhanced HOS cell growth, whereas higher Cu concentrations [i.e., 2.5% and 1% (w/w)] were cytotoxic. The observed effects of Cu concentration on cell growth correlated with the level of HNE production. Therefore, Cu containing BG may represent a useful biomaterial for research and development studies of bone regeneration.