Michael Gelinsky

Functionally graded materials for biomedical applications

Functional gradation is one characteristic feature of living tissue. Bio-inspired materials open new approaches for manufacturing implants for bone replacement. Different routes for new implant materials are presented using the principle of functional gradation. An artificial biomaterial for knee joint replacement has been developed by building a graded structure consisting of ultra-high molecular weight polyethylene (UHMWPE) fibre reinforced high-density polyethylene combined with a surface of UHMWPE. The ingrowth behaviour of titanium implants into hard tissue can be improved by depositing a graded biopolymer coating of fibronectin, collagen types I and III with a gradation, derived from the mechanisms occurring during healing in vivo. Functionally graded porous hydroxyapatite (HAP) ceramics can be produced using alternative routes, e.g. sintering of laminated structures of HAP tapes filled with polymer spheres or combining biodegradable polyesters such as polylactide, polylactide-co-glycolide and polyglycolide, with carbonated nanocrystalline hydroxyapatite. HAP–collagen I scaffolds are an appropriate material for in vitro growth of bone. The scaffold has to be functionally graded in order to create an optimised mechanical behaviour as well as the intended improvement of the cell ingrowth.

Three-dimensional printing of hierarchical and tough mesoporous bioactive glass scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability.

New generation biomaterials for bone regeneration should be highly bioactive, resorbable and mechanically strong. Mesoporous bioactive glass (MBG), a novel bioactive material, has been used to study bone regeneration due to its excellent bioactivity, degradation and drug delivery ability, however, the construction of three-dimensional (3-D) MBG scaffolds (as for other bioactive inorganic scaffolds) for bone regeneration remains a significant challenge due to their inherent brittleness and low strength. In this brief communication we report a new facile method to prepare hierarchical and multifunctional MBG scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability for application in bone regeneration by a modified 3-D printing technique using polyvinylalcohol (PVA) as a binder. The method provides a new way to solve commonly existing issues for inorganic scaffold materials, for example, uncontrollable pore architectures, low strength, high brittleness and the requirement for a second sintering at high temperature. The 3-D printed MBG scaffolds obtained possess a high mechanical strength about 200 times that of traditional polyurethane foam templated MBG scaffolds. They have a highly controllable pore architecture, excellent apatite mineralization ability and sustained drug delivery properties. Our study indicates that 3-D printed MBG scaffolds may be an excellent candidate for bone regeneration.

Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure.

Hyperthermia and local drug delivery have been proposed as potential therapeutic approaches for bone defects resulting from malignant bone tumors. The development of bioactive materials with magnetic and drug delivery properties may potentially meet this target. The aim of this study was to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermic and local drug delivery applications. To this end iron (Fe)-containing MBG (Fe-MBG) scaffolds with a hierarchical large pores structure (300-500 μm) and fingerprint-like mesopores (4.5 nm) have been prepared. The effects of Fe on the mesopore structure and physiochemical, magnetic, drug delivery and biological properties of MBG scaffolds have been systematically investigated. The results show that the morphology of the mesopores varied from straight channels to curved fingerprint-like channels after incorporation of Fe into MBG scaffolds. The magnetism of MBG scaffolds can be tailored by controlling the Fe content. Furthermore, the incorporation of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and the expression of bone-related genes (ALP and OCN) in human bone marrow mesenchymal stem cells (BMSC) attached to the scaffolds. The Fe-MBG scaffolds obtained also possessed high specific surface areas and demonstrated sustained drug delivery. Thus Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunctionality of Fe-MBG scaffolds suggests that there is great potential for their use in the treatment and regeneration of large-bone defects caused by malignant bone tumors through a combination of hyperthermia, local drug delivery and osteoconductivity.

3D-printing of highly uniform CaSiO3 ceramic scaffolds: preparation, characterization and in vivo osteogenesis

Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods could not efficiently control the pore structure and mechanical strength of CS scaffolds, resulting in unstable in vivo osteogenesis. The aim of this study is to set out to solve these problems by applying a modified 3D-printing method to prepare highly uniform CS scaffolds with controllable pore structure and improved mechanical strength. The in vivo osteogenesis of the prepared 3D-printed CS scaffolds was further investigated by implanting them in the femur defects of rats. The results show that the CS scaffolds prepared by the modified 3D-printing method have uniform scaffold morphology. The pore size and pore structure of CS scaffolds can be efficiently adjusted. The compressive strength of 3D-printed CS scaffolds is around 120 times that of conventional polyurethane templated CS scaffolds. 3D-Printed CS scaffolds possess excellent apatite-mineralization ability in simulated body fluids. Micro-CT analysis has shown that 3D-printed CS scaffolds play an important role in assisting the regeneration of bone defects in vivo. The healing level of bone defects implanted by 3D-printed CS scaffolds is obviously higher than that of 3D-printed β-tricalcium phosphate (β-TCP) scaffolds at both 4 and 8 weeks. Hematoxylin and eosin (H&E) staining shows that 3D-printed CS scaffolds induce higher quality of the newly formed bone than 3D-printed β-TCP scaffolds. Immunohistochemical analyses have further shown that stronger expression of human type I collagen (COL1) and alkaline phosphate (ALP) in the bone matrix occurs in the 3D-printed CS scaffolds than in the 3D-printed β-TCP scaffolds. Considering these important advantages, such as controllable structure architecture, significant improvement in mechanical strength, excellent in vivo osteogenesis and since there is no need for second-time sintering, it is indicated that the prepared 3D-printed CS scaffolds are a promising material for application in bone regeneration.

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.

Bioactive SrO–SiO2 glass with well-ordered mesopores: Characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO(2) glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO(2) in an effort to develop a bioactive mesoporous SrO-SiO(2) (Sr-Si) glass with the capacity to deliver Sr(2+) ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr(2+) on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr-Si glass were investigated. The prepared mesoporous Sr-Si glass was found to have an excellent release profile of bioactive Sr(2+) ions and dexamethasone, and the incorporation of Sr(2+) improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr-Si glass had no cytotoxic effects and its release of Sr(2+) and SiO(4)(4-) ions enhanced alkaline phosphatase activity - a marker of osteogenic cell differentiation - in human bone mesenchymal stem cells. Mesoporous Sr-Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr(2+) into mesoporous SiO(2) glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications.

Hierarchical mesoporous bioactive glass/alginate composite scaffolds fabricated by three-dimensional plotting for bone tissue engineering

Constructing bioactive scaffolds with controllable architecture for bone tissue engineering and drug delivery still maintains a significant challenge. In this study, we have developed a composite material consisting of mesoporous bioactive glass (MBG) and concentrated alginate pastes for fabrication of hierarchical scaffolds by 3D plotting. The scaffold structure contains well-ordered nano-channels, micropores as well as controllable macropores beneficial for bone tissue engineering applications and drug delivery. The structural architecture of the scaffolds has been optimized by efficient designing of the plotting coordination. The effects of MBG on mechanical strength, apatite mineralization, cytocompatibility and drug delivery properties of the composite scaffolds have been systematically studied. Transmission electron microscopy, scanning electron microscopy and energy-dispersive spectrometry were used to characterize composition and microstructure of the composite scaffolds. The MBG/alginate pastes showed good processability in the 3D plotting process, in which stable MBG/alginate composite scaffolds with controllable architecture can be prepared. The incorporation of MBG particles significantly improved the mechanical properties and apatite-mineralization ability of alginate scaffolds as well as enhanced the attachment and alkaline phosphatase activity of human bone marrow-derived mesenchymal stem cells cultivated onto the scaffolds. Dexamethasone, used as a model drug, can be efficiently loaded in MBG particles and then incorporated into alginate scaffolds resulting in a more sustained release as a function of the MBG content. Our results have indicated that 3D-plotted MBG incorporated alginate scaffolds with well-ordered nano-pores, controllable large pores, and significantly improved physicochemical, biological and drug-delivery properties could be a platform for bone tissue engineering.

Toxicity of Tungsten Carbide and Cobalt-Doped Tungsten Carbide Nanoparticles in Mammalian Cells in Vitro

Background Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. Objective We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobaltdoped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. Methods We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendro cyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). Results Chemical–physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. Conclusions Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright

Cultivation of human bone marrow stromal cells on three-dimensional scaffolds of mineralized collagen: influence of seeding density on colonization, proliferation and osteogenic differentiation

In this study human bone marrow stromal cells (hBMSCs) were cultured on three‐dimensional porous scaffolds of biomimetically mineralized collagen type I developed for bone engineering. Three different cell numbers were used for seeding of the nanocomposites, and the impact of the seeding density on proliferation and osteogenic differentiation of hBMSCs was investigated. In addition, the effect of the seeding cell number on seeding efficiency and distribution of the cells within the scaffolds was studied. Our data revealed that the open and interconnecting porosity of the mineralized collagen scaffolds allows a very efficient seeding for all seeding densities tested. Although penetration of the cells into the interior of the scaffolds was demonstrated for all seeding densities, the application of higher cell numbers resulted in a better colonization also of the deeper scaffold regions. A substantial influence of the seeding density was observed on proliferation and osteogenic differentiation of hBMSCs. Thus, the highest proliferation rate and specific alkaline phosphatase activity was found for the cell matrix constructs seeded with the lowest density. RT–PCR analyses revealed a higher expression of alkaline phosphatase and bone sialoprotein II at lower seeding densities; however, expression of osteopontin was unaffected by the seeding cell number. Our results demonstrated that the seeding density might be an important factor for the development of optimal cell matrix constructs for bone tissue engineering. Copyright