Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

Biomorphous porous hydroxyapatite-ceramics from rattan (Calamus Rotang).

The three-dimensional, highly oriented pore channel anatomy of native rattan (Calamus rotang) was used as a template to fabricate biomorphous hydroxyapatite (Ca(5)(PO(4))(3)OH) ceramics designed for bone regeneration scaffolds. A low viscous hydroxyapatite-sol was prepared from triethyl phosphite and calcium nitrate tetrahydrate and repeatedly vacuum infiltrated into the native template. The template was subsequently pyrolysed at 800 degrees C to form a biocarbon replica of the native tissue. Heat treatment at 1,300 degrees C in air atmosphere caused oxidation of the carbon skeleton and sintering of the hydroxyapatite. SEM analysis confirmed detailed replication of rattan anatomy. Porosity of the samples measured by mercury porosimetry showed a multimodal pore size distribution in the range of 300 nm to 300 microm. Phase composition was determined by XRD and FT-IR revealing hydroxyapatite as the dominant phase with minimum fractions of CaO and Ca(3)(PO(4))(2). The biomorphous scaffolds with a total porosity of 70-80% obtained a compressive strength of 3-5 MPa in axial direction and 1-2 MPa in radial direction of the pore channel orientation. Bending strength was determined in a coaxial double ring test resulting in a maximum bending strength of approximately 2 MPa.

In vitro response of hFOB cells to pamidronate modified sodium silicate coated cellulose scaffolds.

The aim of the present study was to evaluate the suitability of cellulose-based scaffolds coated with pure sodium silicate gel and sodium silicate gels accumulated with different concentrations of the bisphosphonate pamidronate as scaffolds for attachment, proliferation and differentiation of human fetal osteoblasts (hFOB 1.19). Human osteoblasts were cultured in vitro for a period up to 14 days on different cellulose scaffolds. Unmodified and sodium silicate coated cellulose scaffolds were used as control. Two surface-coated modifications of cellulose were applied. The scaffolds were coated in a modified two-step dip coating process with pure sodium silicate gel and pamidronate enriched sodium silicate gel, respectively. In order to investigate the influence of the pamidronate, concentrations of 0.667 mg Na-pamidronate/ml sodium silicate solution, 0.333 mg Na-pamidronate/ml sodium silicate solution and 3.33 x 10(-3) mg Na-pamidronate/ml sodium silicate solution were used for the coating process. Cell proliferation, vitality and attachment were examined by means of cell counting, WST-1 test, fluorescence and scanning electron microscopy. The relative grade of differentiation of hFOB cells was examined by using quantitative real-time polymerase chain reaction (qRT-PCR) analysis for the gene expression of alkaline phosphatase and osteocalcin. Proliferation and differentiation of human osteoblasts was enhanced by the sodium silicate coatings accumulated with pamidronate compared to pure sodium silicate coatings. There was a reciprocal correlation of vitality with the concentration of pamidronate. The highest vitality was found on surfaces with the lowest pamidronate accumulation. Alkaline phosphatase, an early differentiation marker, was overexpressed after 7 days in cells on all pamidronate-containing surfaces (up to 350% compared to untreated cellulose). Osteocalcin, a late differentiation marker, was overexpressed after 14 days in cells on all coated surfaces (up to 300,000% compared to untreated cellulose). The results indicate that due to the modified coating procedure a homogeneous coating and thus, an enhancement of cell attachment and subsequent cellular functions can be achieved. Low concentrations of pamidronate seem to have a relevant effect on cell proliferation and vitality and, therefore, can be recommended for the improvement of the properties of a biomaterial.

Cellulose-based scaffold materials for cartilage tissue engineering.

Non-woven cellulose II fabrics were used as scaffolds for in vitro cartilage tissue engineering. The scaffolds were activated in a saturated Ca(OH)(2) solution and subsequently coated with a calcium phosphate layer precipitated from a supersaturated physiological solution. Chondrocyte cell response and cartilage development were investigated. The cell adherence was significantly improved compared to untreated cellulose fabrics, and the proliferation and vitality of the adhered chondrocytes were excellent, indicating the biocompatibility of these materials. A homogeneous distribution of the seeded cells was possible and the development of cartilageous tissue could be proved. In contact with a physiological chondrocyte solution, calcium is expected to be leached out from the precipitated layer, which might lead to a microenvironment that triggers the development of cartilage in a way similar to cartilage repair in the vicinity of subchondral bone.

Engineering of vascularized transplantable bone tissues: induction of axial vascularization in an osteoconductive matrix using an arteriovenous loop.

INTRODUCTION: Vascularization remains an obstacle to engineering of larger volume bone tissues. Our aim was to induce axial vascularization in a processed bovine cancellous bone (PBCB) matrix using an arteriovenous (AV) loop (artery, vein graft, and vein). METHODS: Custom-made PBCB discs (9 x 5 mm) were implanted into rats. In group A (n = 19), the matrices were inserted into microsurgically constructed AV loops between the femoral vessels using a vein graft from the contralateral side. In group B (n = 19), there was no vascular carrier. The matrices were encased in isolation chambers. After 2, 4, and 8 weeks, the animals were perfused with India ink via the abdominal aorta. Matrices were explanted and subjected to histological and morphometric analysis. Results were compared with intravital dynamic micro & magnetic resonance imaging and scanning electron microscopy images of vascular corrosion replicas. RESULTS: In group A, significant vascularization of the matrix had occurred by the 8th week. At this time, vascular remodeling with organization into vessels of different sizes was evident. Blood vessels originated from all 3 zones of the AV loop. Group A was significantly superior to group B in terms of vascular density and vascularization kinetics. DISCUSSION: This study demonstrates for the first time successful vascularization of solid porous matrices by means of an AV loop. Injection of osteogenic cells into axially prevascularized matrices may eventually create functional bioartificial bone tissues for reconstruction of large defects.

Hydroxyapatite growth on anodic TiO2 nanotubes.

In the present work, we study the growth of hydroxyapatite formation on different TiO(2) nanotube layers. The nanotube layers were fabricated by electrochemical anodization of titanium in fluoride-containing electrolytes. To study various nanotube lengths, layers with an individual tube diameter of 100 nm were grown to a thickness of approximately 2 mum or 500 nm. The ability to form apatite on the nanotube layers was examined by immersion tests combined with SEM, XRD and FT-IR investigations. For reference, experiments were also carried out on compact anodic TiO(2) layers. The results clearly show that the presence of the nanotubes on a titanium surface enhances the apatite formation and that the 2-mum thick nanotube layer triggers deposition faster than the thinner layers. Tubes annealed to anatase, or a mixture of anatase and rutile are clearly more efficient in promoting apatite formation than the tubes in their "as-formed" amorphous state.

Toughening of dental glass ionomer cements with reactive glass fibres.

A fibre reinforced glass ionomer cement (FRGIC) for dental applications was loaded with 20 vol% short fibres (430 microm) with a glass composition in the system SiO(2)-Al(2)O(3)-CaF(2)-Na(3)AlF(6). The fracture toughness and the total energy release rate were examined. A 20% anisotropic fibre alignment was observed, perpendicular to the loading direction. An increase of fracture toughness of 140% and of total energy release rate of 440% was achieved compared to the unreinforced glass ionomer cement. Matrix-fibre interface reaction is supposed to exert the major influence on mechanical behaviour of FRGIC by controlling fibre pull-out and thus the total energy release rate.

Hydroxyapatite formation on alkali-treated titanium with different content of Na+ in the surface layer.

Titanium can form a bone-like apatite layer on its surface in SBF when it is treated in NaOH. When pre-treated titanium is exposed to SBF, the alkali ions are released from the surface into the surrounding fluid. The Na+ ions increase the degree of supersaturation of the soaking solution with respect to apatite by increasing pH. On the other hand, the released Na+ cause an increase in external alkalinity that triggers an inflammatory response and leads to cell death. Therefore, it would be beneficial to decrease the release of Na+ into the surrounding tissue. The purpose of this study was to evaluate the hydroxyapatite formation on alkali-treated titanium with different content of Na+ in the surface layer. Using SEM, gravimetric analysis and measurement of calcium and phosphate concentration, it was found that the rate of apatite formation was not significantly influenced by a lower amount of Na+ in the surface layer. Titanium with the lowest content of Na+ could be more suitable for implantation in the human body. The amount of alkali ions released in the surrounding tissue is lower and the rate of apatite formation is identical to titanium with the highest content of Na+ in the surface layer.

Mechanical properties and in vitro cell compatibility of hydroxyapatite ceramics with graded pore structure.

In order to improve the mechanical strength of hydroxyapatite (HA) ceramics used as osteoimplants and to enhance cellular penetration functionally graded ceramics with a transition in porosity from the surface towards the centre were designed. The multilayer structures were prepared by multiple tape casting based on an aqueous HA slurry containing polybutylmethacrylate (PBMA) spheres with diameters ranging from 100 to 300 microns. After burning out the PBMA, pores of 70-200 microns were generated. The pore-graded laminates were sintered at temperatures between 1250 degrees C and 1450 degrees C. Bending strength of the pore-graded ceramics was approximately 50% higher as compared to HA of the same pore volume fraction but without gradient structure. The materials were tested in vitro for attachment and activity of osteoblast-like MC3T3-E1 cells over a period of 3 weeks. Cells formed confluent layers on the ceramic surface, penetrated into the graded porosity ranging from 100-150 microns to 250-300 microns in size and showed increasing alkaline phosphatase activity over 3 weeks. The results demonstrated initial in vitro cell compatibility of the functionally graded HA materials and their potential as osteoimplants.

Biomimetic apatite formation on chemically treated titanium.

Titanium treated in NaOH can form hydroxycarbonated apatite (HCA) after exposition in simulated body fluid (SBF). Generally, titanium is covered with a passive oxide layer. In NaOH this passive film dissolves and an amorphous layer containing alkali ions is formed on the surface. When exposed to SBF, the alkali ions are released from the amorphous layer and hydronium ions enter into the surface layer, resulting in the formation of Ti-OH groups in the surface. The released Na(+) ions increase the degree of supersaturation of the soaking solution with respect to apatite by increasing pH, and Ti-OH groups induce apatite nucleation on the titanium surface. The acid etching of titanium in HCl under inert atmosphere was examined as a pretreatment to obtain a uniform initial titanium surface before alkali treatment. Acid etching in HCl leads to the formation of a micro-roughened surface, which remains after alkali treatment in NaOH. It was shown by SEM, gravimetric and solution analysis that the apatite nucleation was uniform and the thickness of precipitated HCA layer increased continuously with time. The treatment of titanium by acid etching in HCl and subsequently in NaOH is a suitable method for providing the metal implant with bone-bonding ability.

Reactive fibre reinforced glass ionomer cements.

The mechanical properties of glass ionomer cements used in restorative dentistry reinforced by chopped glass fibres were investigated. Reactive glass fibres with a composition in the system SiO(2)-Al(2)O(3)-CaF(2)-Na(3)AlF(6) and a thickness of 26 microm were drawn by a bushing process. The manufacturing parameters were optimized with respect to maximum strength of the glass fibre reinforced ionomer cements. Powder to liquid ratio, pre-treatment of the glass, grain size distribution and fibre volume fraction were varied. Glass fibre and cement were characterized by X-ray diffraction, transmission electron microscopy and energy dispersive spectroscopy techniques, respectively. The highest flexural strength of the reinforced cement (15.6 MPa) was found by compounding 20 vol% reactive fibres and extending the initial dry gelation period up to 30 min. Microscopic examination of the fractured cements indicated a distinct reactive layer at the fibre surface. A pronounced fibre pull out mode gives rise to an additional work-of-fracture contributed by pulling the fibres out of the fracture surface.

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.

Cobalt-releasing 1393 bioactive glass-derived scaffolds for bone tissue engineering applications.

Loading biomaterials with angiogenic therapeutics has emerged as a promising approach for developing superior biomaterials for engineering bone constructs. In this context, cobalt-releasing materials are of interest as Co is a known angiogenic agent. In this study, we report on cobalt-releasing three-dimensional (3D) scaffolds based on a silicate bioactive glass. Novel melt-derived "1393" glass (53 wt % SiO2, 6 wt % Na2O, 12 wt % K2O, 5 wt % MgO, 20 wt % CaO, and 4 wt % P2O5) with CoO substituted for CaO was fabricated and was used to produce a 3D porous scaffold by the foam replica technique. Glass structural and thermal properties as well as scaffold macrostructure, compressive strength, acellular bioactivity, and Co release in simulated body fluid (SBF) were investigated. In particular, detailed insights into the physicochemical reactions occurring at the scaffold-fluid interface were derived from advanced micro-particle-induced X-ray emission/Rutherford backscattering spectrometry analysis. CoO is shown to act in a concentration-dependent manner as both a network former and a network modifier. At a concentration of 5 wt % CoO, the glass transition point (Tg) of the glass was reduced because of the replacement of stronger Si-O bonds with Co-O bonds in the glass network. Compressive strengths of >2 MPa were measured for Co-containing 1393-derived scaffolds, which are comparable to values of human spongy bone. SBF studies showed that all glass scaffolds form a calcium phosphate (CaP) layer, and for 1393-1Co and 1393-5Co, CaP layers with incorporated traces of Co were observed. The highest Co concentrations of ∼12 ppm were released in SBF after reaction for 21 days, which are known to be within therapeutic ranges reported for Co(2+) ions.

Thermal-induced residual stresses affect the lifetime of zirconia-veneer crowns.

OBJECTIVES: The purposes of this study were to investigate the effects of thermal residual stresses on the reliability and lifetime of zirconia-veneer crowns. METHODS: One hundred and twenty eight second upper premolar zirconia-veneer crowns were manufactured for testing the initial strength (n=64) and under cyclic fatigue (n=64). Zirconia copings (YZ Cubes, VITA Zahnfabrik, CTE: alphac=10.5 ppm/°C) were milled using a Cerec3 InLab (Sirona) machine and sintered to a final thickness of 0.7 mm. Sixty-four copings were sandblasted with 105 µm alumina particles (15 s, 3 cm distance, 45° angle, 0.4 MPa pressure) in order to trigger a tetragonalmonoclinic transformation and to produce a rough surface. The copings were veneered using two different porcelains (VM9, VITA Zahnfabrik, CTE: alphaVM9=9.1 ppm/°C, Lava Ceram, 3M ESPE, CTE: alphaLava=10.2 ppm/°C) so to result in crowns with either high thermal mismatch (+1.4 ppm/°C with VM9) and low thermal mismatch (+0.3 ppm/°C with Lava Ceram). The porcelains were applied by the same operator and fired (VITA Vacumat 4000) according to the firing schedules defined by the manufacturers to a final thickness of 1.4mm (total crown thickness=2.1mm, core/veneer ratio=0.5). After the last glaze firing the crowns were cooled following a fast (600°C/min) or a slow (30°C/min) cooling protocol. The glazed crowns were submitted to a sliding-motion (0.7 mm lateral movement) cyclic fatigue in a chewing simulator (SD Mechatronik) under 20 kg (~200 N load) weight until failure (chipping) (n=16). The other half of the crowns were subjected to a compressive loading test in an universal testing machine (Instron model 4240) until failure at a cross-head speed of 0.75 mm/min (n=16). The failure probability for initial strength and cyclic fatigue was performed using a Weibull distribution approach at a scale factor of n=16. RESULTS: The compressive strength test showed a low sensitivity to detect reliability variations regarding thermal stresses created within the veneer layer of tested crowns. For cyclic fatigue, slow cooling resulted in statistically higher cycles to failure only for the crowns that presented a high thermal mismatch between core and veneer (VM9 group). Comparisons between veneers with high or low thermal mismatches showed statistically higher sigma0 for Lava Ceram-veneered crowns only when the groups were fast-cooled. SIGNIFICANCE: Minimizing the thermal residual stresses within the veneer through the use of a veneer with a closer CTE to the zirconia delays the failure of zirconia-veneer crowns. Slow-cooling increases the lifetime of crowns presenting large differences in CTE between the zirconia core and the veneering porcelain.

Self-healing materials.

Self-healing materials are able to partially or completely heal damage inflicted on them, e.g., crack formation; it is anticipated that the original functionality can be restored. This article covers the design and generic principles of self-healing materials through a wide range of different material classes including metals, ceramics, concrete, and polymers. Recent key developments and future challenges in the field of self-healing materials are summarised, and generic, fundamental material-independent principles and mechanism are discussed and evaluated.

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 ß-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.

Lifetime prediction of CAD/CAM dental ceramics.

The dynamic fatigue method was used to obtain subcritical crack growth parameters n and A for a commercial feldspathic dental porcelain and for a lanthanum-glass-infiltrated alumina glass ceramic. Five stress rates d sigma/dt ranging from 50 to 0.01 MPa s(-1) were applied. The inert strength values were calculated with the use of Weibull statistics and maximum-likelihood approaches for the Weibull parameter m. Strength-probability-time (SPT) diagrams were derived for both materials. The alumina glass composite showed a high fracture strength sigma(0) (442 MPa) at a failure probability of P(F) = 63.2% and a high resistance against subcritical crack growth (n = 36.5). The development of strength under fatigue conditions was calculated for exemplary 1 year. The strength of the alumina glass material dropped to 228 MPa within this period. This fact is due to a low content of infiltrated lanthanum glass phase in the composite material (25 wt%). In contrast, for the high-silica-glass-containing porcelain a distinct decrease of strength sigma(0) from initial 133 to 47 MPa after 1 year was predicted. This, mainly because of a low crack growth resistance (n = 16.8) of the feldspathic porcelain. Much lower strength values were calculated, assuming a failure probability of P(F) = 5%. The decrease is mainly caused by the sensitivity of high-glass-containing ceramics against water corrosion.