Anja Lode

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His‐239, Cys‐148 or Cys‐152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.

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.

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.

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

Direct Plotting of Three‐Dimensional Hollow Fiber Scaffolds Based on Concentrated Alginate Pastes for Tissue Engineering

Porous scaffolds for tissue engineering applications consisting of hollow alginate fibers are presented. They are prepared using self-made shell/core nozzles and a 3D plotting device. Such materials open up the possibility to generate biodegradable tissue constructs with a preformed vascular system or can act as matrices for engineering of complex organs or 3D tissue models.

In Vitro Evaluation of Textile Chitosan Scaffolds for Tissue Engineering using Human Bone Marrow Stromal Cells

Textile chitosan fiber scaffolds were developed and tested in terms of biocompatibility for human bone marrow stromal cells (hBMSCs). A part of the scaffolds was further modified by coating with fibrillar collagen type I in order to biologize the surface. hBMSCs of two donors were used for cell culture experiments in vitro. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were osteogenically induced after 3 days and cultivated for up to 28 days on the scaffolds. Activity of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) was analyzed to evaluate proliferation as well as osteogenic differentiation. We found a 3.5-6-fold increase in the cell number, whereas the collagen coating did not noticeably influence these factors. Osteogenic differentiation was confirmed by the course of ALP activity and immunostaining of osteocalcin. The feature of the collagen-coated as well as the raw chitosan fiber scaffolds to support attachment, proliferation, and differentiation of hBMSCs suggests a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

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

Alginate/Nanohydroxyapatite Scaffolds with Designed Core/Shell Structures Fabricated by 3D Plotting and in Situ Mineralization for Bone Tissue Engineering

Composite scaffolds, especially polymer/hydroxyapatite (HAP) composite scaffolds with predesigned structures, are promising materials for bone tissue engineering. Various methods including direct mixing of HAP powder with polymers or incubating polymer scaffolds in simulated body fluid for preparing polymer/HAP composite scaffolds are either uncontrolled or require long times of incubation. In this work, alginate/nano-HAP composite scaffolds with designed pore parameters and core/shell structures were fabricated using 3D plotting technique and in situ mineralization under mild conditions (at room temperature and without the use of any organic solvents). Light microscopy, scanning electron microscopy, microcomputer tomography, X-ray diffraction, and Fourier transform infrared spectroscopy were applied to characterize the fabricated scaffolds. Mechanical properties and protein delivery of the scaffolds were evaluated, as well as the cell response to the scaffolds by culturing human bone-marrow-derived mesenchymal stem cells (hBMSC). The obtained data indicate that this method is suitable to fabricate alginate/nano-HAP composite scaffolds with a layer of nano-HAP, coating the surface of the alginate strands homogeneously and completely. The surface mineralization enhanced the mechanical properties and improved the cell attachment and spreading, as well as supported sustaining protein release, compared to pure alginate scaffolds without nano-HAP shell layer. The results demonstrated that the method provides an interesting option for bone tissue engineering application.

Fabrication and characterization of regenerated silk scaffolds reinforced with natural silk fibers for bone tissue engineering

We introduce a novel Bombyx mori silk-based composite material developed for bone tissue engineering. Three-dimensional scaffolds were fabricated by embedding of natural degummed silk fibers in a matrix of regenerated fibroin, followed by freeze-drying. Different ratios of fibers to fibroin were investigated with respect to their influence on mechanical and biological properties. For all scaffold types, an interconnected porous structure suitable for cell penetration was proven by scanning electron microscopy. Compressive tests, carried out in static and cyclic mode under dry as well as wet conditions, revealed a strong impact of fiber reinforcement on compressive modulus and compressive stress. Cell culture experiments with human mesenchymal stem cells demonstrated that the fiber/fibroin composite scaffolds support cell attachment, proliferation, as well as differentiation along the osteoblastic lineage. Considering the excellent mechanical and biological properties, novel fiber/fibroin scaffolds appear to be an interesting structure for prospect studies in bone tissue engineering.