Electric field-assisted formation of organically modified hydroxyapatite (ormoHAP) spheres in carboxymethylated gelatin gels.

UNLABELLED: A biomimetic strategy was developed in order to prepare organically modified hydroxyapatite (ormoHAP) with spherical shape. The technical approach is based on electric field-assisted migration of calcium ions and phosphate ions into a hydrogel composed of carboxymethylated gelatin. The electric field as well as the carboxymethylation using glucuronic acid (GlcA) significantly accelerates the mineralization process, which makes the process feasible for lab scale production of ormoHAP spheres and probably beyond. A further process was developed for gentle separation of the ormoHAP spheres from the gelatin gel without compromising the morphology of the mineral. The term ormoHAP was chosen since morphological analyses using electron microscopy (SEM, TEM) and element analysis (EDX, FT-IR, XRD) confirmed that carboxymethylated gelatin molecules use to act as organic templates for the formation of nanocrystalline HAP. The hydroxyapatite (HAP) crystals self-organize to form hollow spheres with diameters ranging from 100 to 500nm. The combination of the biocompatible chemical composition and the unique structure of the nanocomposites is considered to be a useful basis for future applications in functionalized degradable biomaterials. STATEMENT OF SIGNIFICANCE: A novel bioinspired mineralization process was developed based on electric field-assisted migration of calcium and phosphate ions into biochemically carboxymethylated gelatin acting as organic template. Advantages over conventional hydroxyapatite include particle size distribution and homogeneity as well as achievable mechanical properties of relevant composites. Moreover, specifically developed calcium ion or phosphate ion release during degradation can be useful to adjust the fate of bone cells in order to manipulate remodeling processes. The hollow structure of the spheres can be useful for embedding drugs in the core, encapsulated by the highly mineralized outer shell. In this way, controlled drug release could be achieved, which enables advanced strategies for threating bone-related diseases, e.g. osteoporosis and multiple myeloma.

Artificial extracellular matrices of collagen and sulphated hyaluronan enhance the differentiation of human mesenchymal stem cells in the presence of dexamethasone.

In this study we investigated the potential of artificial extracellular matrix (aECM) coatings containing collagen II and two types of glycosaminoglycan (GAGs) with different degrees of sulphation to promote human bone formation in biomedical applications. To this end their impact on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assessed. The cell proliferation was found to be significantly retarded in the first 14 days of culture on surfaces coated with collagen II and GAGs (coll-II/GAG) as compared to tissue culture polystyrol (TCPS) and those coated with collagen II. At later time points it only tended to be retarded on coll-II/sHya3.1. Heat-inactivation of the serum significantly reduced cell numbers on collagen II and coll-II/sHya3.1. Alkaline phosphatase (ALP) activity and calcium deposition, on the other hand, were higher for coatings containing sHya3.1 and were not significantly changed by heat-inactivation of the serum. Expression levels of the bone matrix proteins bone sialoprotein (BSP-II) and osteopontin (OP) were also increased on aECM coatings as compared to TCPS, which further validated the differentiation of hMSCs towards the osteogenic lineage. These observations reveal that aECM coatings, in particular those containing sHya3.1, are suitable to promote the osteogenic differentiation of hMSCs.

Calcium phosphate phases integrated in silica/collagen nanocomposite xerogels enhance the bioactivity and ultimately manipulate the osteoblast/osteoclast ratio in a human co-culture model.

A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes respectively, was used to characterize the influence of biomaterial modification on the bioactivity and ultimately the ratio of bone-forming to bone-resorbing cells cultivated directly on the surface. Nanocomposites of silica and collagen have been shown to function as skeletal structures in nature and were reproduced in vitro by using a sol-gel approach. The resulting xerogels exhibit a number of features that make it a valuable system for the development of innovative materials for bone substitution applications. In the present study, the incorporation of different calcium phosphate phases in silica/collagen-based gels was demonstrated to enhance the bioactivity of these samples. This ability of the biomaterial to precipitate calcium phosphate on the surface when incubated in simulated body fluids or cell culture medium is generally considered to an advantageous property for bone substitution materials. By co-cultivating human osteoblasts and osteoclasts up to 42 days on the xerogels, we demonstrate that the long-term ratio of these cell types depends on the level of bioactivity of the substrate samples. Biphasic silica/collagen xerogels exhibited comparably low bioactivity but encouraged proliferation of osteoblasts in comparison to osteoclast formation. A balanced ratio of both cell types was detected for moderately bioactive triphasic xerogels with 5% calcium phosphate. However, enhancing the bioactivity of the xerogel samples by increasing the calcium phosphate phase percentage to 20% resulted in a diminished number of osteoblasts in favor of osteoclast formation. Quantitative evaluation was carried out by biochemical methods (calcium, DNA, ALP, TRAP 5b) as well as RT-PCR (ALP, BSP II, OC, RANKL, TRAP, CALCR, VTNR, CTSK), and was supported by confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP) as well as scanning electron microscopy.

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-ß1 (TGF-ß1).

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-ß1 (TGF-ß1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-ß1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.

[Resorbable bone substitution materials: An overview of commercially available materials and new approaches in the field of composites]. / Resorbierbare Knochenersatzmaterialien: Eine Übersicht kommerziell verfügbarer Werkstoffe und neuer Forschungsansätze auf dem Gebiet der Komposite.

When acquired or inborn bony defects cannot heal by the natural regeneration process due to being above the critical size or to particular diseases, e.g. osteoporosis, it becomes necessary to use bone substitute materials. These are materials which replace the missing bone tissue in host tissue and stimulate the bone healing process by mechanical and structural support either alone or in combination with other substances. This supporting effect can be attended by natural as well as artificial bone substitute materials and in a variety of ways. The biological efficiency of a bone substitute material is often classified with respect to the terms osteogenic, osteoconductive and osteoinductive stimulation. In reality however there is an overlap of several effective principles. Due to the limited availability of autologous bone and the disadvantages for the patient associated with the removal, intensive research is being carried out into artificial alternatives. The present article aims to offer some orientation in this confusing field by a systematic description of the various bone substitute materials.

Development of an osteoblast/osteoclast co-culture derived by human bone marrow stromal cells and human monocytes for biomaterials testing.

The communication of bone-forming osteoblasts and bone-resorbing osteoclasts is a fundamental requirement for balanced bone remodelling. For biomaterial research, development of in vitro models is necessary to investigate this communication. In the present study human bone marrow stromal cells and human monocytes were cultivated in order to differentiate into osteoblasts and osteoclasts, respectively. Finally, a cultivation regime was identified which firstly induces the differentiation of the human bone marrow stromal cells followed by the induction of osteoclastogenesis through the osteoblasts formed--without the external addition of the factors RANKL and M-CSF. As a feedback on osteoblasts enhanced gene expression of BSP II was detected for modifications which facilitated the formation of large multinuclear osteoclasts. Phenotype characterization was performed by biochemical methods (DNA, LDH, ALP, TRAP 5b), gene expression analysis (ALP, BSP II, RANKL, IL-6, VTNR, CTSK, TRAP, OSCAR, CALCR) as well as light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. After establishing this model on polystyrene, similar positive results were obtained for cultivation on a relevant bone substitution material--a composite xerogel of silica, collagen, and calcium phosphate.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: Biomimetic potential and applications.

In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part I. Isolation and identification of chitin.

Marine invertebrate organisms including sponges (Porifera) not only provide an abundant source of biologically active secondary metabolites but also inspire investigations to develop biomimetic composites, scaffolds and templates for practical use in materials science, biomedicine and tissue engineering. Here, we presented a detailed study of the structural and physico-chemical properties of three-dimensional skeletal scaffolds of the marine sponges Aiolochroia crassa, Aplysina aerophoba, A. cauliformis, A. cavernicola, and A. fulva (Verongida: Demospongiae). We show that these fibrous scaffolds have a multilayered design and are made of chitin. (13)C solid-state NMR spectroscopy, NEXAFS, and IR spectroscopy as well as chitinase digestion and test were applied in order to unequivocally prove the existence of alpha-chitin in all investigated species.

In vitro osteoclastogenesis on textile chitosan scaffold.

Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) for up to 18 days. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP), acidic compartments using 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP), immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR). The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption--one main requirement for successful bone tissue engineering.

How is wettability of titanium surfaces influenced by their preparation and storage conditions?

The effect of two different etching procedures with inorganic acids (HSE and CSE)-one using additionally strongly oxidising conditions due to the presence of CrO(3) (CSE)-and consecutive storage conditions (dry methanol and air) for previous corundum blasted titanium surfaces is compared with respect to their wettability behaviour and the potential of the etching processes for removing remaining blasting material. The etching procedures result in distinct different surface morphologies. Whereas the HSE surface shows sub-mm to sub-mum structures but neither porosity nor undercuts, the CSE surface is extremely rugged and porous with structures protruding the more homogeneously attacked areas by several micrometers. By EDX analysis both remaining blasting material and chromium and sulphur from the etching treatment has been detected on the CSE surfaces only. Both surfaces states show super-hydrophilic behaviour immediately after etching and storage up to 28 days in dry methanol. Whereas contact with air does not change super-hydrophilicity for the CSE samples, wettings angles of the HSE samples increase within minutes and reach about angles of about 60 degrees and 90 degrees after one and 2 days exposure to air, respectively. The increasing hydrophobicity is discussed with respect to the formation of a surface coverage from hydrocarbons originating from aromatic compounds present in traces in air.

Fibrils of different collagen types containing immobilised proteoglycans (PGs) as coatings: characterisation and influence on osteoblast behaviour.

Collagen, the main organic component of bone, is used as a coating on titanium implants and as a scaffold material in bone tissue engineering. Surface modifications of titanium which promote osteoblast adhesion, proliferation and synthesis of collagen by osteoblasts are desirable. One biomimetic approach is the coating of titanium with collagen in fibrillar form. Other organic components of bone may be bound to fibrils and exert additional effects. In this study, the collagen types I-III were compared regarding their ability to bind the proteoglycans decorin and biglycan, which are found in bone. More collagen was bound to collagen II fibrils than to those of types I and III. Therefore, titanium surfaces were coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the effect of the proteoglycans on human primary osteoblast behaviour. In addition, the growth factor TGF-beta1 was adsorbed onto surfaces coated with fibrils of collagen type II containing biglycan or decorin or neither to investigate the influence of decorin and biglycan on the effect of TGF-beta1 on osteoblasts. Fibril-bound biglycan and decorin influence primary osteoblast behaviour by themselves. The presence of substrate-bound biglycan or decorin influences the effect of TGF-beta1. These results may be important when designing collagen-based coatings or scaffolds for tissue engineering, including those loaded with growth factors.

Adjusting the chlorhexidine content of calcium phosphate coatings by electrochemically assisted co-deposition from aqueous solutions.

Currently, a number of strategies to create either biologically active or antimicrobial surfaces of biomaterials are being developed and commercially applied. However, for metallic implants in contact with bone, both osteomyelitis and a fast and stable long-term fixation of implants are challenges to be overcome, especially in the case of bad bone quality. Therefore, the present work aims to develop compound coatings of calcium phosphate phases (CPP) and chlorhexidine (CHD) that combine bioactive properties with a strategy to prevent initial bacterial adhesion and thus offer a possible solution to the two major problems of implant surgery mentioned above. Using electrochemically assisted deposition of CPP on samples of Ti6Al4V together with the pH-dependent solubility of CHD, the preparation of coatings with a wide range of CHD concentrations (150 ng/cm(2) to 65 microg/cm(2)) from electrolytes with CHD concentrations between 50 and 200 microM was possible, thus allowing the adaptation of implant surface properties to different surgical and patient situations. Detailed SEM and FTIR analysis showed that coatings are formed by a co-deposition process of both phases and that CHD interacts with the deposition and transformation of CPP in the coating. For high CHD contents, coatings consist of CHD crystals coated by nano-crystalline hydroxyapatite.

Surface modification of titanium-based alloys with bioactive molecules using electrochemically fixed nucleic acids.

A new method of surface modification for titanium (alloys) with bioactive molecules was developed with the intention of providing a new basis of implant adaptation for particular requirements of certain medical indications. Nucleic acid single strands are fixed electrochemically via their termini (regiospecifically) by growing an oxide layer on Ti6Al7Nb anodically. It could be shown that they are accessible to subsequent hybridization with complementary strands at physiological pH. Amount of nucleic acids immobilized and hybridized were determined radioanalytically using 32P-labelled nucleic acids. Stable fixation was attained at and above potentials of 4 V(SCE). Up to 4 pmol/cm2 of nucleic acid single strands could be immobilized and hybridization efficiencies up to 1.0 were reached. Hybridization efficiency was found to depend on surface density of immobilized oligonucleotides, while hybridization rates increased when MgCl2 was added. A conjugate consisting of an oligonucleotide complementary to the immobilized strand and the hexapeptide GRGDSP with RGD as an integrin recognition site was synthesized. This conjugate was able to bind to integrins on osteoblasts. It was shown that this conjugate binds to the anchor strand fixed on Ti6Al7Nb to an extent comparable with the unconjugated complementary strand.

Comparison of microfocus- and synchrotron X-ray tomography for the analysis of osteointegration around Ti6Al4V implants.

Micro-computed tomography (microCT) provides quantitative three-dimensional information of bone around titanium implants similar to classical histology. The study, based on an animal model, containing cuboid-shaped biofunctionalised Ti6Al4V implants with surrounding bone after 4 weeks, is performed using 3 microCT-systems with X-ray tubes, one synchrotron-radiation-based microCT-system (SRmicroCT), and classical histology. Although the spatial resolution of the microCT-systems is comparable, only the results of SRmicroCT agree with results of classical histology. The X-ray tube sources give rise to huge artefacts in the tomograms (interface scattering, beam hardening), which impaired the quantitative analysis of bone up to about 200microm from the implant surface. Due to the non-destructive character of microCT the specimens can be subsequently examined by classical histology without restriction. The quantitative comparison of bone formation uncovers the strong dependence of the newly formed bone from the selected slice. This implies the necessity of 3D analysis. SRmicroCT and classical histology prove that surface modifications of the titanium implant significantly influence the bone formation. Using SRmicroCT, the preparation artefacts due to cutting and polishing are excluded.

Transforming growth factor beta1 immobilized adsorptively on Ti6Al4V and collagen type I coated Ti6Al4V maintains its biological activity.

Titanium and titanium alloys are often used for orthopedic and dental implants. Osseointegration of Ti6Al4V may be improved not only by precoating of the surface with extracellular matrix proteins like collagen type I but also by additional immobilization of growth factors. In the present study, transforming growth factor beta1 (TGF-beta1) which is known as an inducer of collagen synthesis was immobilized adsorptively on uncoated and collagen type I coated Ti6Al4V surfaces. TGF-beta1 was found immobilized slightly faster to collagen type I coated than to uncoated Ti6Al4V and released slower from the collagen coated material. Immobilized TGF-beta1 is biologically active for at least 3 weeks storage at 4 degrees C. Sterilization by ethylene oxide inactivates immobilized TGF-beta1. In osteoblasts cultured on implants with adsorptively immobilized TGF-beta1, mRNA level and specific catalytic activity of alkaline phosphatase as well as accumulation of calcium and phosphate were found reduced, whereas procollagen alpha1(I) mRNA level and the rate of collagen synthesis were increased.

Electrochemically assisted deposition of thin calcium phosphate coatings at near-physiological pH and temperature.

An electrochemical method for the deposition of calcium phosphate phases on titanium surfaces using the galvanostatic mode is presented. Deposition was performed in a (Ca(2+) / H(x)PO(4) ((3-x)-))-containing electrolyte near physiological conditions with regard to pH (6.4) and temperature (36 degrees C). Cathodic alkalization leads first to the formation of a thin homogeneous layer that shows a nanoscale surface topography of alternating wall-like elevations and channels. It is thought that these channels in the calcium phosphate prelayer are formed as pathways for hydroxyl ions and hydrogen. Upon this layer, spheres of amorphous calcium phosphate (ACP) are formed as indicated by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy. According to transmission electron microscopy images, these spheres consist of small clusters of calcium phosphate (approximately 30 nm) and can grow up to 300 nm in diameter. Characteristic for this ACP is a high water content as seen by FTIR. As a function of current density, the ACP is then transformed into crystalline hydroxyapatite (HAP), which was identified using FTIR and X-ray diffraction. The morphology of the HAP crystals can be described as needles with dimensions of <500-nm length and <60-nm width. By choice of different electrochemical parameters, a homogeneous coating of either ACP, HAP, or the intermediate phase can be achieved, as shown in a kinetic phase diagram, thus allowing the formation of coatings with different properties in solubility and morphology.

Proliferation and differentiation of rat calvarial osteoblasts on type I collagen-coated titanium alloy.

Several attempts have been made to improve osseointegration of titanium alloy as an implant material by modification of its surface. In the present study, proliferation, differentiation, and mineralization of osteoblasts on type I collagen-coated Ti6Al4V were investigated. The activity of alkaline phosphatase and the accumulation of calcium by osteoblasts grown on titanium alloy were significantly higher compared to cells grown on polystyrene. Precoating of the implant surface with type I collagen did not extensively affect proliferation, the activity of alkaline phosphatase, collagen synthesis, calcium accumulation, or the mRNA levels for collagen I alpha1, osteopontin, osteocalcin, MMP-2, and TIMP-2. Maximum collagen synthesis by osteoblasts was observed at day 4 of culture independent of the type of implant material. The specific activity of alkaline phosphatase reached its maximum at day 18 of culture. Accumulation of calcium and elevated mRNA levels for osteocalcin were found at day 22. These results indicate that collagen-coating alone is not sufficient to accelerate differentiation of rat calvarial osteoblasts on Ti6Al4V.

Electrochemical behavior of titanium-based materials - are there relations to biocompatibility?

For biomedical applications the physico-chemical properties of oxide layers, always present in titanium-based materials, are of special interest because the biological system is in direct contact only with these oxides. Using electrochemical impedance spectroscopy and galvanostatic polarization it is shown that the different compositions of c.p.-titanium, Ti6Al4V, and Ti6Al7Nb result in different physico-chemical properties of air formed passive layers and anodic oxide layers. This may have a direct impact on the biocompatibility of these materials. Results of impedance spectroscopy distinctly differ in the flatband potentials as well as in the donor densities of air-formed passive layers with Ti6Al7Nb showing an approximately 50% smaller donor density than the other materials. Anodic galvanostatic polarization results in voltage-charge density curves with distinct differences in the Faraday efficiency epsilon of the oxide formation between Ti6Al7Nb and c.p.-titanium/Ti6Al4V, especially for low current densities. These effects correlate strongly with the donor densities in the air formed passive films of the examined materials. SEM-images of anodic oxide layers show a blister containing surface morphology of the outer part of the oxide layers for all materials. This morphology is probably caused by oxygen evolution, a process which relies on the transfer of electrons through the growing anodic oxide layers and strongly depends on the donor density in the air formed passive layers. Again, the much more pronounced morphology on c.p. titanium/Ti6Al4V agrees with the different donor densities in the air formed passive layers on the materials. These findings correlate with the good biocompatibility of Ti6Al7Nb and suggest that conduction mechanisms, in air formed passive layers and anodic oxide layers, contribute to processes that determine the biocompatibility of these materials.

Titanium powder sintering for preparation of a porous functionally graded material destined for orthopaedic implants.

This work focuses on basic research into a P/M processed, porous-surfaced and functionally graded material (FGM) destined for a permanent skeletal replacement implant with improved structural compatibility. Based on a perpendicular gradient in porosity the Young's modulus of the material is adapted to the elastic properties of bone in order to prevent stress shielding effects and to provide better long-term performance of the implant-bone system. Using coarse Ti particle fractions the sintering process was accelerated by silicon-assisted liquid-phase sintering (LPS) resulting in a substantial improvement of the neck geometry. A novel evaluation for the strength of the sinter contacts was proposed. The Young's modulus of uniform non-graded stacks ranged from 5 to 80 GPa as determined by ultrasound velocity measurements. Thus, the typical range for cortical bone (10-29 GPa) was covered. The magnitude of the Poisson's ratio proved to be distinctly dependent on the porosity. Specimens with porosity gradients were successfully fabricated and characterized using quantitative description of the microstructural geometry and acoustic microscopy.

Biomimetic coatings functionalized with adhesion peptides for dental implants.

A complete biological integration into the surrounding tissues (bone, gingiva) is a critical step for clinical success of a dental implant. In this work biomimetic coatings consisting either of collagen type I (for the gingiva region) and hydroxyapatite (HAP) or mineralized collagen (for the bone interface) have been developed as suitable surfaces regarding the interfaces. Additionally, using these biomimetic coatings as a matrix, adhesion peptides were bound to further increase the specificity of titanium implant surfaces. To enhance cell attachment in the gingiva region, a linear adhesion peptide developed from a laminin sequence (TWYKIAFQRNRK) was bound to collagen, whereas for the bone interface, a cyclic RGD peptide was bound to HAP and mineralized collagen using adequate anchor systems. The biological potential of these coatings deduced from cell attachment experiments with HaCaT human keratinocytes and MC3T3-E1 mouse osteoblasts showed the best results for collagen and laminin sequence coating for the gingiva region and mineralized collagen and RGD peptide coatings for regions with bone contact. Our concept opens promising approaches to improve the biological integration of dental implants.