Chemical nanoroughening of Ti40Nb surfaces and its effect on human mesenchymal stromal cell response.

Samples of low modulus beta-type Ti40Nb and cp2-Ti were chemically treated with 98% H2 SO4 + 30% H2 O2 (vol. ratio 1:1) solution. Surface analytical studies conducted with HR-SEM, AFM, and XPS identified a characteristic nanoroughness of the alloy surface related with a network of nanopits of ∼25 nm diameter. This is very similar to that obtained for cp2-Ti. The treatment enhances the oxide layer growth compared to mechanically ground states and causes a strong enrichment of Nb2 O5 relative to TiO2 on the alloy surface. The in vitro analyses clearly indicated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity, and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP). Surface structures which were generated mimic the cytoplasmic projections of the cells on the nanoscale. Those effects are more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions is discussed.

Sulfated hyaluronan/collagen I matrices enhance the osteogenic differentiation of human mesenchymal stromal cells in vitro even in the absence of dexamethasone.

Glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix (ECM) involved in different steps of the regulation of cellular differentiation. In this study artificial extracellular matrices (aECM) consisting of collagen (Col) I and different GAG derivatives were used as a substrate for human mesenchymal stromal cells (hMSC) to study osteogenic differentiation in vitro. hMSC were cultured on aECM containing col and hyaluronan sulfates (HyaS) with increasing degrees of sulfation (DS(S)) and were compared with aECM containing col and the natural GAG hyaluronan or chondroitin 4-sulfate. hMSC were analyzed for osteogenic differentiation markers such as calcium phosphate deposition, tissue non-specific alkaline phosphatase (TNAP) and expression of runt-related transcription factor 2 (runx2), osteocalcin (ocn) and bone sialoprotein II (bspII). Compared with aECM containing Col and natural GAG all Col/HyaS-containing aECM induced an increase in calcium phosphate deposition, TNAP activity and tnap expression. These effects were also seen in the absence of dexamethasone (an established osteogenic supplement). The expression of runx2 and ocn was not altered and the expression of bspII was diminished on the col/HyaS-containing aECM. The impact of the Col/HyaS-containing aECM on hMSC differentiation was independent of the DS(S) of the HyaS derivatives, indicating the importance of the primary (C-6) hydroxyl group of N-acetylglucosamine. These results suggest that Col/HyaS-containing aECM are able to stimulate hMSC to undergo osteogenic differentiation even in the absence of dexamethasone, which makes these matrices an interesting tool for hMSC-based tissue engineering applications and biomaterial functionalizations to enhance bone formation.

The impact of heat treatment on interactions of contact-poled biphasic calcium phosphates with proteins and cells.

A number of studies have reported improved bone integration for calcium phosphate based materials electrically "poled" by an external electric field prior to implantation. In our study we investigated the effects of electrical polarization of a biphasic ceramic composed of 80% hydroxyapatite and 20% ß-tricalcium phosphate. As contact poling involves elevated temperatures as a prerequisite for inducing charge, we used two reference types: samples without any heat treatment and poling, and samples with no poling but heat treatment identical to that of the poled samples. All heat-treated samples (poled or unpoled) showed an improved wettability, which was attributed to a reduced hydrocarbon contamination. Heat treatment alone provoked an accelerated spreading of osteoblast-like cells, whereas on poled samples a retarded cell spreading was observed. While proliferation and several differentiation markers were not influenced by either heat treatment or poling, the release of proinflammatory cytokines interleukin-6 and -8 was significantly reduced for all heat-treated samples, irrespective of additional electrical poling. The study demonstrated that the behaviour of cells in contact with poled biphasic ceramics was influenced by two parameters: heating and charge. Our data revealed that heating of the calcium phosphate ceramics had a much more pronounced effect on cell behaviour than charge.

Nuclear factor of activated T cells mediates oxidised LDL-induced calcification of vascular smooth muscle cells.

AIMS/HYPOTHESIS: Vascular calcification is a prominent feature of both atherosclerosis and diabetes, and is clinically associated with osteoporosis. The expression of bone-regulatory factors and the impact of oxidative stress in aortic calcification are well-documented. Recently, nuclear factor of activated T cells (NFAT) cytoplasmic, calcineurin-dependent 1 (NFATc1) was identified in calcified aortic valves and has been implicated in vascular calcification. Therefore, we assessed the mechanisms of osteogenic transdifferentiation of vascular smooth muscle cells induced by oxidised LDL (oxLDL) and evaluated the role of NFAT in this process. METHODS: Human coronary artery smooth muscle cells (HCASMCs) were cultured for 21 days in medium supplemented with oxLDL. NFAT was inhibited using the NFAT inhibitor VIVIT, or by knockdown with small interfering RNA (siRNA). Osteogenic transdifferentiation was assessed by gene expression, matrix mineralisation and alkaline phosphatase activity. RESULTS: Exposure to oxLDL caused the transformation of HCASMCs towards an osteoblast-like phenotype based on increased mineral matrix formation and RUNX2 expression. NFATc1 blockade completely prevented oxLDL-induced osteogenic transformation of HCASMCs as well as oxLDL-induced stimulation of osteoblast differentiation. In contrast, matrix mineralisation induced by osteogenic medium was independent of the NFAT pathway. Of note, oxLDL-conditioned medium from HCASMCs transferred to bone cells promoted osteoblast mineralisation. Consistent with these in vitro findings, diabetic rats with a twofold increase in oxidised lipid levels displayed higher aortic calcium concentrations and increased expression of osteogenic markers and production of NFATc1. CONCLUSIONS/INTERPRETATION: Our results identify the NFAT signalling pathway as a novel regulator of oxLDL-induced transdifferentiation of vascular smooth muscle cells towards an osteoblast-like phenotype.

In vitro osteogenic potential of human bone marrow stromal cells cultivated in porous scaffolds from mineralized collagen.

Porous 3D structures from mineralized collagen were fabricated applying a procedure in which collagen fibril reassembly and precipitation of nanocrystalline hydroxyapatite (HA) occur simultaneously. The resulting matrices were evaluated in vitro with respect to their suitability as scaffolds for bone tissue engineering. We found a high capacity of the material to bind serum proteins as well as to absorb Ca2+ ions, which could be advantageous to promote cell attachment, growth, and differentiation. Human bone marrow stromal cells (hBMSCs) were seeded onto the 3D scaffolds and cultivated for 4 weeks in the presence and absence of osteogenic supplements. We studied viability, proliferation, and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content, and alkaline phosphatase (ALP) activity. Furthermore, the expression for bone-related genes (ALP, bone sialo protein II (BSP II), and osteocalcin) was analyzed. In our investigation we found a 2.5-fold to 5-fold raise in DNA content and an increase of ALP activity for osteogenic induced hBMSC on collagen HA scaffolds. The expression of ALP and BSP II in these cells was also stimulated in the course of cultivation; however, we did not detect an upregulation of osteocalcin gene expression. These data suggest, that porous collagen HA scaffolds are suitable for the expansion and osteogenic differentiation of hBMSC and are therefore promising candidates for application as bone grafts.

Cathepsin C gene variants in aggressive periodontitis.

Cathepsin C (CTSC) mutations are known to cause Papillon-Lefèvre syndrome. The aim of this study was to examine the association of CTSC genotype with susceptibility to non-syndromic aggressive periodontitis. The CTSC gene was analyzed in 110 persons with generalized aggressive periodontitis in comparison with 78 control individuals, after identifying different variants in a cohort of 100 persons. Five out of 19 discovered variants were included in this association study, representing 5 single-nucleotide polymorphism groups in tight linkage disequilibrium. The relevance of genotypes on enzyme function was examined. The carrier frequency of the missense variant p.I453V was significantly increased in persons with disease compared with healthy control individuals (17.3% vs. 6.4%, p < 0.05). CTSC activity in leukocytes from individuals harboring this variant was significantly reduced (119.8 Delta OD/min*10(5) cells, 95% confidence interval 17.4-174.9, p = 0.018). No influence of promoter variants was found on mRNA expression. The results support the hypothesis that CTSC gene variants contribute to increased susceptibility in generalized aggressive periodontitis.

[A 42-year-old farmer with nonspecific leucocytosis and elevated transaminases. Acute septic reaction in Coxiella burnetii infection]. / 42-jähriger Landwirt mit unklarer Leukozytose und erhöhten Transaminasen. Akute septische Reaction bei Coxiella-burnetii Infektion.

We report about a 42-year-old farmer with leucocytosis, elevation of transaminases and liver cirrhosis as an underlying condition. The diagnosis of Q fever hepatitis was made through liver biopsy and serology. Under therapy with doxycycline, transaminases initially increased again; after switching to ciprofloxacin, the patient could be discharged 3 weeks after admission. Q fever is caused by Coxiella burnetii. The most frequent acute manifestation is a self-limiting flu-like illness. Chronic Q fever mostly presents as endocarditis. The diagnosis is made through histology ("doughnut" granulomas), PCR, serology (acute: anti-phase II antibodies, chronic: anti-phase I antibodies) and culture. Standard therapy is doxycycline.

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.

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.

Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid.

In this study, the proliferation and differentiation of rat calvarial osteoblasts cultured on either (1) calcium-phosphate bone cement Biocement D, (2) Biocement D with 2.5% (w/w) mineralized collagen type I, or (3) Biocement D with 2.5% (w/w) mineralized collagen type I and 3% (w/w) citric acid were investigated. Incubation of the composites in cell-culture medium resulted in a fast decrease of pH and calcium concentration as well as in an increase of phosphate concentration. Although these effects occurred with all investigated materials, the lowest extent could be observed for the citric-acid-containing composites. As shown by scanning-electron microscopy, osteoblasts adhered to the composite surfaces. Proliferation and differentiation of the cells grown on the composites were found to be reduced compared to cells grown on tissue-culture polystyrene. Cells cultured in the vicinity of the composites but without direct contact also exhibited a reduced rate of proliferation, reduced alkaline phosphatase activity, and reduced mineralization. Simulating the changes in calcium and phosphate concentration occasioned by the composites through exposing cells to EGTA and phosphate gives rise to the same effects of reducing proliferation, ALP activity, and mineralization. No indication for apoptosis in cells exposed to low calcium and high phosphate concentrations was found. The number of necrotic cells, however, increased after incubation with EGTA and phosphate. For assessment of cell-composite interactions and the success of the composites in vivo, as well as for more effective material development, it seems to be important to know how changes in microenvironmental pH and ion composition of the material affect cellular proliferation and differentiation.

Phosphoserine--a convenient compound for modification of calcium phosphate bone cement collagen composites.

Temporary bone replacement materials on the basis of calcium phosphates and hydroxyapatite (HAP) are used in surgery for filling bone defects. Components which are able to control the nucleation and crystal growth of HAP through their functional groups and which can additionally activate bone cells may be helpful in the development of materials with enhanced remodelling in vivo. In this study, the influence of O-phospho-L-serine (PS) on the materials properties of calcium phosphate bone cement composites was investigated. For up to an addition of 25 mg/g PS a strong increase in the stability of the cements under load was determined. The material was studied by scanning electron microscopy and transmission electron microscopy. A more dense microstructure and a plate-like morphology of the HAP-crystals were detected in the modified composites compared with the non-modified samples. By X-ray powder diffraction an inhibition of the dissolution of alpha-tricalcium phosphate (alpha-TCP) and dicalciumphosphate anhydrous (DCPA) particles was found. alpha-TCP and DCPA are the main constituents of the cement precursor. The results of cell culture studies using rat calvaria osteoblasts demonstrate a good viability of the cells on the PS-modified material. Furthermore, the proliferation and differentiation were found to be enhanced on the PS-modified material.

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.

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.

Collagen type I-coating of Ti6Al4V promotes adhesion of osteoblasts.

The initial contact of osteoblasts with implant surfaces is an important event for osseointegration of implants. Osseointegration of Ti6Al4V may be improved by precoating of its surface with collagen type I. In this study, the adhesion of rat calvarial osteoblasts to uncoated and collagen type I-coated titanium alloy was investigated over a period of 24 h. Collagen type I-coating accelerates initial adhesion of osteoblasts in the presence of fetal calf serum. One hour after plating, no differences in the percentage of adherent cells between the surfaces investigated were found. Adhesion of osteoblasts to uncoated surfaces was reduced by the GRGDSP peptide by about 70%, whereas adhesion to collagen type I-coated surfaces remained unaffected by treatment of the cells with the peptide. Cell adhesion to coated materials was reduced by about 80% by anti-integrin beta1 antibody. The integrin beta1 antibody did not influence the adhesion to uncoated titanium alloy. The results suggest that osteoblasts adhere to collagen type I-coated materials via integrin beta1 but not by interacting with RGD peptides, whereas adhesion to uncoated titanium alloy is mediated by RGD sequences but not via integrin beta1. Fibronectin does not seem to be involved in the adhesion of osteoblasts to either coated or uncoated titanium alloy.

Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia.

This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats. All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining. In newborn rats, only caveolin-1 was found in the hyaline cartilage. Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures. Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes. These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes. There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation.

Ultrastructural identification of caveolae and immunocytochemical as well as biochemical detection of caveolin in chondrocytes.

Using fluorescence immunocytochemistry, transmission electron microscopy and Western blotting, we have shown that caveolae and caveolin are abundant on chondrocytes of different cartilaginous structures of newborn and adult rat knee joints. Caveolin was detected in chondrocytes of the outer layer of articular cartilage, in the fibrocartilage of the menisci, and in fibrocartilage-like cells at tendon and ligament insertions. Electron microscopical studies revealed caveolae-like invaginations along the plasmalemmal membrane of articular chondrocytes and fibrocartilage cells. Immunoblot analysis demonstrated caveolin in detergent-insoluble and soluble complexes isolated from cultured rat chondrocytes.

Prostaglandin E2 affects differently the release of inflammatory mediators from resident macrophages by LPS and muramyl tripeptides.

LPS and MTP-PE (liposome-encapsulated N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-:[1',2'dipalmitoyl -sni-glycero-3-(hydroxy-phosphoryl-oxyl)] etylamide) induce in liver macrophages a synthesis and release of TNF-alpha, nitric oxide and prostanoids. Both agents induce an expression of mRNA's encoding TNF-alpha, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and of corresponding proteins. LPS and MTP-PE induce a rapid activation of the extracellular regulated kinase (ERK) isoenzymes-1 and -2. Inhibition of map kinase isoenzymes leads to a decreased release of TNF-alpha, nitric oxide and prostaglandin (PG) E2 after both agents. The transcription factors NF-kappaB and AP-1 are strongly activated by LPS within 30 minutes. MTP-PE induces a weak activation of both transcription factors only after 5 hours. Inhibition of NF-kappaB inhibits the LPS- but not the MTP-PE-induced release of TNF-alpha, nitric oxide and PGE2. PGE2 release after LPS is higher than after MTP-PE. Exogenously added PGE2 inhibits the activation of map kinase and TNF-alpha release by LPS, but not by MTP-PE. Release of nitric oxide after LPS and MTP-PE is enhanced after prior addition of PGE2. PGD2 is without any effect. MTP-PE, but not LPS, induces a cytotoxicity of Kupffer cells against P815 tumor target cells. The MTP-PE-induced cytotoxicity is reduced by TNF-alpha neutralizing antibodies, indicating the involvement of TNF-alpha. Thus our results suggest that the different potencies of LPS and MTP-PE as immunomodulators probably result from different actions on Kupffer cells, resulting in differences in the amounts and kinetics of released TNF-alpha and PGE2, and that PGE2 plays an important regulatory role in the action of LPS, but not in the actions of MTP-PE.

Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism.

In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1.