3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization.

Bone substitute fabrication is of interest to meet the worldwide incidence of bone disorders. Physical chitosan hydrogels with intertwined apatite particles were chosen to meet the bio-physical and mechanical properties required by a potential bone substitute. A set up for 3-D printing by robocasting was found adequate to fabricate scaffolds. Inks consisted of suspensions of calcium phosphate particles in chitosan acidic aqueous solution. The inks are shear-thinning and consist of a suspension of dispersed platelet aggregates of dicalcium phosphate dihydrate in a continuous chitosan phase. The rheological properties of the inks were studied, including their shear-thinning characteristics and yield stress. Scaffolds were printed in basic water/ethanol baths to induce transformation of chitosan-calcium phosphates suspension into physical hydrogel of chitosan mineralized with apatite. Scaffolds consisted of a chitosan polymeric matrix intertwined with poorly crystalline apatite particles. Results indicate that ink rheological properties could be tuned by controlling ink composition: in particular, more printable inks are obtained with higher chitosan concentration (0.19 mol·L-1).

Designing smart particles for the assembly of complex macroscopic structures.

Particle get-together: Surface functionalization with a branched copolymer surfactant is used to create responsive inorganic particles that can self-assemble in complex structures. The assembly process is triggered by a pH switch that reversibly activates multiple hydrogen bonds between ceramic particles (see picture; yellow) and soft templates (n-decane; green).

Nanotechnology approaches to improve dental implants.

The requirements imposed by the enormous scale and overall complexity of designing new implants or complete organ regeneration are well beyond the reach of present technology in many dimensions, including nanoscale, as researchers do not yet have the basic knowledge required to achieve these goals. The need for a synthetic implant to address multiple physical and biologic factors imposes tremendous constraints on the choice of suitable materials. There is a strong belief that nanoscale materials will produce a new generation of implant materials with high efficiency, low cost, and high volume. The nanoscale in materials processing is truly a new frontier. Metallic dental implants have been used successfully for decades, but they have serious shortcomings related to their osseointegration and the fact that their mechanical properties do not match those of bone. This paper reviews recent advances in the fabrication of novel coatings and nanopatterning of dental implants. It also provides a general summary of the state of the art in dental implant science and describes possible advantages of nanotechnology for future improvements. The ultimate goal is to produce materials and therapies that will bring state-of-the-art technology to the bedside and improve quality of life and current standards of care.

Three-dimensional biomimetic mineralization of dense hydrogel templates.

An electric-current-assisted method was used to mineralize dense hydrogels and create hydroxyapatite/hydrogel composites with unique hierarchical structures. The microstructure of the final material can be controlled by the mineralization technique and the chemistry of the organic matrix. A hydroxyapatite/hydrogel composite was obtained with a large inorganic content (approximately 60% of the weight of the organics). After being heated to 1050 degrees C, the sintered inorganic phase has a very uniformly distributed porosity and its Brunauer-Emmett-Teller (BET) surface area is 0.68 m(2)/g.

SEM evaluation of resin-carious dentin interfaces formed by two dentin adhesive systems.

OBJECTIVES: We investigated the influence of dentin tubule direction and identifiable zone of carious dentin on the microstructure and the thickness of the hybrid-like layer (HL) formed by self-etch and etch-rinse adhesive systems. METHODS: An etch-rinse and a self-etching adhesive were bonded to dentin carious zones divided into groups with parallel or perpendicular orientation relative to the dentin tubules at the resin-carious dentin interface (N=5/variable). Bonds were prepared to each of the four zones of carious dentin apparent after staining with Caries Detector: pink, light pink, transparent and apparently normal; six non-carious third molars were controls. The microstructure and thickness of the HL were determined by SEM and compared using three-way ANOVA and Tukey's multiple comparisons (p<0.05). RESULTS: Etch-rinse controls gave thicker HLs than self-etching systems; orientation did not affect thickness for the self-etch system. Perpendicular orientations gave thicker HLs than parallel for the pink zone bonded with the etch-rinse system. For both adhesives, HL thickness in the pink zone was significantly greater than in light pink for the perpendicular group, but no significant differences were found among other variables. HL microstructure was more granular and rougher for the etch-rinse than for the self-etching system. Pores and cracks were obvious in the more demineralized zones. Resin tags were shorter and irregular in the transparent zone and often were completely absent in the outer demineralized zones (pink, light pink). SIGNIFICANCE: Microstructure of bonded interfaces varies markedly depending on adhesive system, tubule orientation and carious zone.

Stress-corrosion crack growth of Si-Na-K-Mg-Ca-P-O bioactive glasses in simulated human physiological environment.

This paper describes research on the stress-corrosion crack growth (SCCG) behavior of a new series of bioactive glasses designed to fabricate coatings on Ti and Co-Cr-based implant alloys. These glasses should provide improved implant fixation between implant and exhibit good mechanical stability in vivo. It is then important to develop an understanding of the mechanisms that control environmentally assisted crack growth in this new family of glasses and its effect on their reliability. Several compositions have been tested in both static and cyclic loading in simulated body fluid. These show only small dependences of SCCG behavior on the composition. Traditional SCCG mechanisms for silicate glasses appear to be operative for the new bioactive glasses studied here. At higher velocities, hydrodynamic effects reduce growth rates under conditions that would rarely pertain for small natural flaws in devices.

Fracture modes under uniaxial compression in hydroxyapatite scaffolds fabricated by robocasting.

The fracture modes of hydroxyapatite (HA) scaffolds fabricated by direct-write assembly (robocasting) are analyzed in this work. Concentrated HA inks with suitable viscoelastic properties were developed to enable the fabrication of prototype structures consisting of a 3-D square mesh of interpenetrating rods. The fracture behavior of these model scaffolds under compressive stresses is determined from in situ uniaxial tests performed in two different directions: perpendicular to the rods and along one of the rod directions. The results are analyzed in terms of the stress field calculated by finite element modeling (FEM). This analysis provides valuable insight into the mechanical behavior of scaffolds for bone tissue engineering applications fabricated by robocasting.

Bioactive glass coatings affect the behavior of osteoblast-like cells.

Functionally graded coatings (FGCs) of bioactive glass on titanium alloy (Ti6Al4V) were fabricated by the enameling technique. These innovative coatings may be an alternative to plasma-sprayed, hydroxyapatite-coated implants. Previously we determined that a preconditioning treatment in simulated body fluid (SBF) helped to stabilize FGCs [Foppiano S et al. Acta Biomater 2006;2(2):133-42]. The primary goal of this work was to assess the in vitro cytocompatibility of preconditioned FGCs with MC3T3-E1.4 mouse pre-osteoblastic cells. We evaluated cell adhesion, proliferation and mineralization on FGCs in comparison to uncoated Ti6Al4V and tissue culture polystyrene (TCPS). No difference in cell adhesion was identified, whereas proliferation was significantly different on all materials, being highest on FGCs followed by TCPS and Ti6Al4V. Qualitative and quantitative mineralization assays indicated that mineralization occurred on all materials. The amount of inorganic phosphate released by the mineralizing layers was significantly different, being highest on TCPS, followed by FGC and uncoated Ti6Al4V. The secondary objective of this work was to assess the ability of the FGCs to affect gene expression, indirectly, by means of their dissolution products, which was assessed by real-time reverse-transcription polymerase chain reaction. The FGC dissolution products induced a 2-fold increase in the expression of Runx-2, and a 20% decrease in the expression of collagen type 1 with respect to TCPS extract. These genes are regulators of osteoblast differentiation and mineralization, respectively. The findings of this study indicate that preconditioned FGCs are cytocompatible and suggest that future work may allow composition changes to induce preferred gene expression.

Sintering and robocasting of beta-tricalcium phosphate scaffolds for orthopaedic applications.

beta-Tricalcium phosphate (beta-TCP) scaffolds with designed, three-dimensional (3-D) geometry and mesoscale porosity have been fabricated by direct-write assembly (robocasting) techniques. Concentrated beta-TCP inks with suitable viscoelastic properties were developed to enable the fabrication of the complex 3-D structures. A comprehensive study of the sintering behavior of TCP as a function of the calcium content in the starting powder was also carried out, and the optimal heat treatment for fabricating scaffolds with dense beta-TCP rods has been determined. Such analysis provides clues to controlling the microstructure of the fabricated structures and, therefore, enabling the fabrication by robocasting of TCP scaffolds with tailored performance for bone tissue engineering applications.

Functionally graded bioactive coatings: reproducibility and stability of the coating under cell culture conditions.

This work sought to provide a basic protocol for treatment of functionally graded bioactive glass coatings (FGC) that reliably adhere to titanium alloy (Ti6Al4V) prior to in vivo evaluation. The effect of the fabrication process on glass structure and reproducibility of the coating's properties, and the effect of cell culture conditions on the integrity of the coating were assessed. The structure of FGCs was compared to that of the as cast glass used as a top coating. X-ray diffraction (XRD) showed that the fabrication process resulted in 5.9+/-3.0 vol.% crystallization, while glass as cast was amorphous. Glass as cast and coatings behaved similarly in simulated body fluid (SBF): an amorphous layer rich in phosphate formed, and it crystallized, over 4 weeks, into apatite-like mineral (Fourier transform infrared spectroscopy (FTIR), XRD, scanning electron microscopy (SEM)). Reproducibility of the fabrication process was tested from three batches of coatings by measuring thickness and crystallinity. MC3T3-E1.4 mouse pre-osteoblast cells were cultured and induced to mineralize on FGCs, either as made or pre-conditioned in SBF. The sub-surface glass silica network in FGCs was compromised by cell culture conditions. A crystalline phosphate was formed during pre-conditioning (XRD, FTIR, and SEM). SBF-pre-conditioning stabilized the coatings. Thus incubation in SBF is recommended to produce a stable coating.

Nanotechnology for dental implants.

With the advent of nanotechnology, an opportunity exists for the engineering of new dental implant materials. Metallic dental implants have been successfully used for decades, but they have shortcomings related to osseointegration and mechanical properties that do not match those of bone. Absent the development of an entirely new class of materials, faster osseointegration of currently available dental implants can be accomplished by various surface modifications. To date, there is no consensus regarding the preferred method(s) of implant surface modification, and further development will be required before the ideal implant surface can be created, let alone become available for clinical use. Current approaches can generally be categorized into three areas: ceramic coatings, surface functionalization, and patterning on the micro- to nanoscale. The distinctions among these are imprecise, as some or all of these approaches can be combined to improve in vivo implant performance. These surface improvements have resulted in durable implants with a high percentage of success and long-term function. Nanotechnology has provided another set of opportunities for the manipulation of implant surfaces in its capacity to mimic the surface topography formed by extracellular matrix components of natural tissue. The possibilities introduced by nanotechnology now permit the tailoring of implant chemistry and structure with an unprecedented degree of control. For the first time, tools are available that can be used to manipulate the physicochemical environment and monitor key cellular events at the molecular level. These new tools and capabilities will result in faster bone formation, reduced healing time, and rapid recovery to function.

Direct ink writing of highly porous and strong glass scaffolds for load-bearing bone defects repair and regeneration.

The quest for synthetic materials to repair load-bearing bone lost because of trauma, cancer, or congenital bone defects requires the development of porous, high-performance scaffolds with exceptional mechanical strength. However, the low mechanical strength of porous bioactive ceramic and glass scaffolds, compared with that of human cortical bone, has limited their use for these applications. In the present work bioactive 6P53B glass scaffolds with superior mechanical strength were fabricated using a direct ink writing technique. The rheological properties of Pluronic® F-127 (referred to hereafter simply as F-127) hydrogel-based inks were optimized for the printing of features as fine as 30 µm and of three-dimensional scaffolds. The mechanical strength and in vitro degradation of the scaffolds were assessed in a simulated body fluid (SBF). The sintered glass scaffolds showed a compressive strength (136 ± 22 MPa) comparable with that of human cortical bone (100-150 MPa), while the porosity (60%) was in the range of that of trabecular bone (50-90%). The strength is ~100-times that of polymer scaffolds and 4-5-times that of ceramic and glass scaffolds with comparable porosities. Despite the strength decrease resulting from weight loss during immersion in SBF, the value (77 MPa) is still far above that of trabecular bone after 3 weeks. The ability to create both porous and strong structures opens a new avenue for fabricating scaffolds for load-bearing bone defect repair and regeneration.

The ionic products of bioactive glass particle dissolution enhance periodontal ligament fibroblast osteocalcin expression and enhance early mineralized tissue development.

This study resulted in enhanced collagen type 1 and osteocalcin expression in human periodontal ligament fibroblasts (hPDLF) when exposed to bioactive glass conditioned media that subsequently may promote early mineralized tissue development. Commercial Bioglass™ (45S5) and experimental bioactive coating glass (6P53-b), were used to make a glass conditioned media (GCM) for comparison to control medium. ICP-MS analysis showed increased concentrations of Ca(2+), PO(4) (3-), Si(4+), and Na(+), for 45S5 GCM and Mg(2+), K(+), Ca(2+), PO(4)(3-), Si(4+), and Na(+) for 6P53-b GCM (relative to control medium). Differentiating hPDLF cultures exposed to 45S5 and 6P53-b GCM showed enhanced expression of collagen type 1 (Col1α1, Col1α2), osteocalcin, and alkaline phosphatase gene expression. These GCM also enhanced osteocalcin protein expression. After 16 d of culture, 45S5 and 6P53-b GCM treated cells showed regions of deep red Alizarin staining, indicating increased Ca within their respective extracellular matrices (ECM), while control-treated cells did not exhibit these features. SEM analysis showed more developed ECM in GCM treated cultures, indicated by multiple tissue layering and abundant collagen fiber bundle formation, while control treated cells did not exhibit these features. SEM analysis showed polygonal structures suggestive of CaP in 45S5 GCM treated cultures. These results indicate the osteogenic potential of bioactive coating glass in periodontal bone defect filling applications.

Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications.

The design of synthetic bone grafts that mimic the structure and composition of bone and possess good surgical handling characteristics remains a major challenge. We report the development of poly(2-hydroxyethyl methacrylate) (pHEMA)-hydroxyapatite (HA) composites termed "FlexBone" that possess osteoconductive mineral content approximating that of human bone yet exhibit elastomeric properties enabling the press-fitting into a defect site. The approach involves crosslinking pHEMA hydrogel in the presence of HA using viscous ethylene glycol as a solvent. The composites exhibit excellent structural integration between the apatite mineral component and the hydroxylated hydrogel matrix. The stiffness of the composite and the ability to withstand compressive stress correlate with the microstructure and content of the mineral component. The incorporation of porous aggregates of HA nanocrystals rather than compact micrometer-sized calcined HA effectively improved the resistance of the composite to crack propagation under compression. Freeze-dried FlexBone containing 50 wt % porous HA nanocrystals could withstand hundreds-of-megapascals compressive stress and >80% compressive strain without exhibiting brittle fractures. Upon equilibration with water, FlexBone retained good structural integration and withstood repetitive moderate (megapascals) compressive stress at body temperature. When subcutaneously implanted in rats, FlexBone supported osteoblastic differentiation of the bone marrow stromal cells pre-seeded on FlexBone. Taken together, the combination of high osteoconductive mineral content, excellent organic-inorganic structural integration, elasticity, and the ability to support osteoblastic differentiation in vivo makes FlexBone a promising candidate for orthopedic applications.

Mechanical properties of calcium phosphate scaffolds fabricated by robocasting.

The mechanical behavior under compressive stresses of beta-tricalcium phosphate (beta-TCP) and hydroxyapatite (HA) scaffolds fabricated by direct-write assembly (robocasting) technique is analyzed. Concentrated colloidal inks prepared from beta-TCP and HA commercial powders were used to fabricate porous structures consisting of a 3-D tetragonal mesh of interpenetrating ceramic rods. The compressive strength and elastic modulus of these model scaffolds were determined by uniaxial testing to compare the relative performance of the selected materials. The effect of a 3-week immersion in simulated body fluid (SBF) on the strength of the scaffolds was also analyzed. The results are compared with those reported in the literature for calcium phosphate scaffolds and human bone. The robocast calcium phosphate scaffolds were found to exhibit excellent mechanical performances in terms of strength, especially the HA structures after SBF immersion, indicating a great potential of this type of scaffolds for use in load-bearing bone tissue engineering applications.

Defect induced asymmetric pit formation on hydroxyapatite.

Defect sites on bone minerals play a critical role in bone remodeling processes. We investigated single crystal hydroxyapatite (100) surfaces bearing crystal defects under acidic dissolution conditions using real-time in situ atomic force microscopy. At defect sites, surface structure-dependent asymmetric hexagonal etch pits were formed, which dominated the overall dissolution rate. Meanwhile, dissolution from the flat terraces proceeded by stochastic formation of flat bottom etch pits. The resulting pit shapes were intrinsically dictated by the HAP crystal structure. Computational modeling also predicted different step energies associated with different facets of the asymmetric etch pits. Our microscopic observations of HAP dissolution are significant for understanding the effects of local surface structure on the bone mineral remodeling process and provide useful insights for the design of novel therapies for treating osteoporosis and dental caries.

Freezing as a path to build complex composites.

Materials that are strong, ultralightweight, and tough are in demand for a range of applications, requiring architectures and components carefully designed from the micrometer down to the nanometer scale. Nacre, a structure found in many molluscan shells, and bone are frequently used as examples for how nature achieves this through hybrid organic-inorganic composites. Unfortunately, it has proven extremely difficult to transcribe nacre-like clever designs into synthetic materials, partly because their intricate structures need to be replicated at several length scales. We demonstrate how the physics of ice formation can be used to develop sophisticated porous and layered-hybrid materials, including artificial bone, ceramic-metal composites, and porous scaffolds for osseous tissue regeneration with strengths up to four times higher than those of materials currently used for implantation.

Atomic dynamics and Marangoni films during liquid-metal spreading.

Despite its apparent simplicity, spreading of liquid metals at high temperatures has defied description and generalization. Wetting at high temperature is usually accompanied by interdiffusion and chemical reaction, but the forces that drive reactive spreading and the mechanisms that control its kinetics have been very poorly understood. The unsolved challenge has been to link macroscopic measurements such as the dynamic contact angle or the speed of a moving liquid front to phenomena occurring at the microscopic and even atomic level in the vicinity of the triple solid-liquid-vapour junction. We have taken a big step towards meeting this challenge. Our systematic analysis of the spreading of metal-metal systems with varying degrees of mutual solubility allows us to report on the fundamental differences between the mechanisms controlling spreading of organic liquids and liquid metals and on formation of Marangoni films driven by surface-tension gradients in high-temperature systems.

A new approach to mineralization of biocompatible hydrogel scaffolds: an efficient process toward 3-dimensional bonelike composites.

As a first step toward the design and fabrication of biomimetic bonelike composite materials, we have developed a template-driven nucleation and mineral growth process for the high-affinity integration of hydroxyapatite with a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel scaffold. A mineralization technique was developed that exposes carboxylate groups on the surface of cross-linked pHEMA, promoting high-affinity nucleation and growth of calcium phosphate on the surface, along with extensive calcification of the hydrogel interior. Robust surface mineral layers a few microns thick were obtained. The same mineralization technique, when applied to a hydrogel that is less prone to surface hydrolysis, led to distinctly different mineralization patterns, in terms of both the extent of mineralization and the crystallinity of the apatite grown on the hydrogel surface. This template-driven mineralization technique provides an efficient approach toward bonelike composites with high mineral-hydrogel interfacial adhesion strength.