Conrado Aparicio

A self-assembly pathway to aligned monodomain gels

Aggregates of charged amphiphilic molecules have been found to access a structure at elevated temperature that templates alignment of supramolecular fibrils over macroscopic scales. The thermal pathway leads to a lamellar plaque structure with fibrous texture that breaks upon cooling into large arrays of aligned nanoscale fibres and forms a strongly birefringent liquid. By manually dragging this liquid crystal from a pipette onto salty media, it is possible to extend this alignment over centimetres in noodle-shaped viscoelastic strings. Using this approach, the solution of supramolecular filaments can be mixed with cells at physiological temperatures to form monodomain gels of aligned cells and filaments. The nature of the self-assembly process and its biocompatibility would allow formation of cellular wires in situ that have any length and customized peptide compositions for use in biological applications.

Corrosion behaviour of commercially pure titanium shot blasted with different materials and sizes of shot particles for dental implant applications

It is well known that the osseointegration of the commercially pure titanium (c.p. Ti) dental implant is improved when the metal is shot blasted in order to increase its surface roughness. This roughness is colonised by bone, which improves implant fixation. However, shot blasting also changes the chemical composition of the implant surface because some shot particles remain adhered on the metal. The c.p. Ti surfaces shot blasted with different materials and sizes of shot particles were tested in order to determine their topographical features (surface roughness, real surface area and the percentage of surface covered by the adhered shot particles) and electrochemical behaviour (open circuit potential, electrochemical impedance spectroscopy and cyclic polarisation). The results demonstrate that the increased surface area of the material because of the increasing surface roughness is not the only cause for differences found in the electrochemical behaviour and corrosion resistance of the blasted c.p. Ti. Among other possible causes, those differences may be attributed to the compressive residual surface stresses induced by shot blasting. All the materials tested have an adequate corrosion and electrochemical behaviour in terms of its possible use as dental implant material.

Bone Regeneration Mediated by Biomimetic Mineralization of a Nanofiber Matrix

Rapid bone regeneration within a three-dimensional defect without the use of bone grafts, exogenous growth factors, or cells remains a major challenge. We report here on the use of self-assembling peptide nanostructured gels to promote bone regeneration that have the capacity to mineralize in biomimetic fashion. The main molecular design was the use of phosphoserine residues in the sequence of a peptide amphiphile known to nucleate hydroxyapatite crystals on the surfaces of nanofibers. We tested the system in a rat femoral critical-size defect by placing pre-assembled nanofiber gels in a 5mm gap and analyzed bone formation with micro-computed tomography and histology. We found within 4 weeks significantly higher bone formation relative to controls lacking phosphorylated residues and comparable bone formation to that observed in animals treated with a clinically used allogenic bone matrix.

In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests

We report on the in vivo histological and mechanical performance of titanium dental implants with a new surface treatment (2Step) consisting of an initial grit-blasting process to produce a micro-rough surface, followed by a combined chemical and thermal treatment that produces a potentially bioactive surface, i.e., that can form an apatitic layer when exposed to biomimetic conditions in vitro. Our aim was to assess the short- and mid-term bone regenerative potential and mechanical retention of 2Step implants in mandible and maxilla of minipigs and compare them with micro-rough grit-blasted, micro-rough acid-etched, and smooth as-machined titanium implants. The percent of bone-to-implant contact after 2, 4, 6, and 10 weeks of implantation as well as the mechanical retention after 4, and 6 weeks of implantation were evaluated with histometric and pull-out tests, respectively, as a measure of the osseointegration of the implants. We also aimed to assess the bioactive nature of 2Step surfaces in vivo. Our results demonstrated that the 2Step treatment produced micro-rough and bioactive implants that accelerated bone tissue regeneration and increased mechanical retention in the bone bed at short periods of implantation in comparison with all other implants tested. This was mostly attributed to the ability of 2Step implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate (a) bone nucleation directly on the implant surface, and (b) bone growing from the implant surface. We also proved that roughness values of Ra≈4.5 μm favoured osseointegration of dental implants at short- and mid-term healing periods, as grit-blasted implants and 2Step implants had higher retention values than as machined and acid-etched implants. The surface quality resulting from the 2Step treatment applied on cpTi provided dental implants with a unique combination of rapid bone regeneration and high mechanical retention.

Development of a Biodegradable Composite Scaffold for Bone Tissue Engineering: Physicochemical, Topographical, Mechanical, Degradation, and Biological Properties

The development of synthetic materials and their use in tissue engineering applications hasattracted much attention in recent years as an option for trabecular bone grafting. Bioabsorbablepolyesters of the poly(α-hydroxy acids) family, and specifically polylactic acid (PLA), are wellknown bioabsorbable materials and are currently used for numerous biomedical applications. The incorporationof an inorganic phase, such as a soluble calcium phosphate glass in the P2O5 − CaO − Na2O − TiO2system, into the polymeric matrix enhances the mechanical integrity of the material. In fact, theflexural elastic modulus increases from 3.2 to 10 GPa with 50 wt/wt % of glass particles.It also improves the biological behavior and modifies the degradation pattern of the polymer. Thepresence of glass particles accelerates the material degradation and induces the formation of calciumphosphate precipitates in the surface of the composite. Therefore, the combination of a bioabsorbablepolymer such as PLA with a soluble calcium phosphate glass leads to a fully degradable compositematerial with a high bone regenerative potential. The success of a 3D scaffold dependson several parameters that go from the macro- to the nanoscale. The solvent and casting technique,together with particulate leaching, allows the elaboration of 95 %-porosity scaffolds with a wellinterconnected macro- and microporosity. Factors such as surface chemistry, surface energy, and topographycan highly affect the cell-material response. Indeed, the addition of glass particles in the PLAmatrix modifies the material surface properties such as wettability AI (Area index or real-surface-area/nominal-arearatio) and roughness, improving the cell response and inducing morphological changes in the cytoskeletonof the osteoblasts. This study offers valuable insight into the parameters affecting cell-scaffoldbehavior, and discusses the special relevance that a comprehensive characterization and manufacturingcontrol of the composite surface can have for monitoring the biological–synthetic interactions.

Spatial organization of osteoblast fibronectin matrix on titanium surfaces: Effects of roughness, chemical heterogeneity and surface energy

We investigated the early events of bone matrix formation, and specifically the role of fibronectin (FN) in the initial osteoblast interaction and the subsequent organization of a provisional FN matrix on different rough titanium (Ti) surfaces. Fluorescein isothiocyanate-labelled FN was preadsorbed on these surfaces and studied for its three-dimensional (3-D) organization by confocal microscopy, while its amount was quantified after NaOH extraction. An irregular pattern of adsorption with a higher amount of protein on topographic peaks than on valleys was observed and attributed to the physicochemical heterogeneity of the rough Ti surfaces. MG63 osteoblast-like cells were further cultured on FN-preadsorbed Ti surfaces and an improved initial cellular interaction was observed with increasing roughness. 3-D reconstruction of the immunofluorescence images after 4 days of incubation revealed that osteoblasts deposit FN fibrils in a specific facet-like pattern that is organized within the secreted total matrix overlying the top of the samples. The thickness of this FN layer increased when the roughness of the underlying topography was increased, but not by more than half of the total maximum peak-to-valley distance, as demonstrated with images showing simultaneous reconstruction of fluorescence and topography after 7 days of cell culture.

Growth of bioactive surfaces on titanium and its alloys for orthopaedic and dental implants

A simple chemical method was established for inducing bioactivity of titanium and its alloys. Recently, T. Kokubo demonstrated that an in vitro chemical-deposited bone-like apatite on Ti with bone-bonding ability could be induced. Following treatment, a dense bone-like apatite layer is formed on the surface of the titanium in simulated body fluid (SBF). Observation of the samples in wet state by means of the environmental scanning electron microscope (ESEM) enabled us to observe the calcium phosphate deposition process in situ over a number of days. One of the most important features of the study is that it was carried out on a single, unchanged titanium sample and the process was not at any stage interrupted. Moreover, it was demonstrated that human osteoblast adhesion and differentiation behaviour are better in bioactive titanium than in the titanium without the chemical treatment.

Bio-inspired stable antimicrobial peptide coatings for dental applications.

We developed a novel titanium coating that has applications for preventing infection-related implant failures in dentistry and orthopedics. The coating incorporates an antimicrobial peptide, GL13K, derived from parotid secretory protein, which has been previously shown to be bactericidal and bacteriostatic in solution. We characterized the resulting physicochemical properties, resistance to degradation, activity against Porphyromonas gingivalis and in vitro cytocompatibility. Porphyromonas gingivalis is a pathogen associated with dental peri-implantitis, an inflammatory response to bacteria resulting in bone loss and implant failure. Our surface modifications obtained a homogeneous, highly hydrophobic and strongly anchored GL13K coating that was resistant to mechanical, thermochemical and enzymatic degradation. The GL13K coatings had a bactericidal effect and thus significantly reduced the number of viable bacteria compared to control surfaces. Finally, adequate proliferation of osteoblasts and human gingival fibroblasts demonstrated the GL13K coatings cytocompatibility. The robustness, antimicrobial activity and cytocompatibility of GL13K-biofunctionalized titanium make it a promising candidate for sustained inhibition of bacterial biofilm growth. This surface chemistry provides a basis for development of multifunctional bioactive surfaces to reduce patient morbidities and improve long-term clinical efficacy of metallic dental and orthopedic implants.

Human-osteoblast proliferation and differentiation on grit-blasted and bioactive titanium for dental applications

Physico-chemical and topographical surface quality of commercially pure titanium (c.p. Ti) dental implants is one of the most influencing factors in the improvement of their osseointegration. In this sense, previously, a two-step method (2S) for obtaining bioactive blasted-rough titanium surfaces was developed for improving short-term (due to its bioactivity) and long-term (due to its roughness) osseointegration. This 2S-method consists of: (1) Grit blasting on titanium surface in order to roughen it, and (2) thermo-chemical (TCh) treatment in order to obtain a bioactive surface with bone-bonding ability. The aim of the present work is to evaluate the in vitro human-osteoblast response (proliferation, differentiation – ALP activity- and cell morphology-studied by environmental scanning electron microscopy) of rough c.p. Ti (grit blasted), bioactive c.p. Ti (thermo-chemically treated) and rough-bioactive c.p. Ti (2S-treated). Different grit materials (Al2O3 and SiC) have been used in order to investigate their influence. The results showed that cell adhesion was statistically higher for the rough and bioactive surfaces, whatever the grit used. Cells proliferated very well on all the c.p. Ti surfaces. If comparing groups with and without TCh (all other treatments being equal) the ALP was always higher in the groups with TCh, indicating stimulation of osteoblast differentiation because of TCh, more significantlly in the groups that were first blasted. Those ALP results were accompanied by a decrease in the value of proliferation, which shows the good behavior of the cells. This results suggest that a rough and bioactive-titanium surface obtained by 2S-treatment enhances adhesion and differentiation activity of human osteoblasts cells.

Biomimetic Mineralization of Woven Bone-Like Nanocomposites: Role of Collagen Cross-Links

Ideal biomaterials for bone grafts must be biocompatible, osteoconductive, osteoinductive and have appropriate mechanical properties. For this, the development of synthetic bone substitutes mimicking natural bone is desirable, but this requires controllable mineralization of the collagen matrix. In this study, densified collagen films (up to 100 μm thick) were fabricated by a plastic compression technique and cross-linked using carbodiimide. Then, collagen-hydroxyapatite composites were prepared by using a polymer-induced liquid-precursor (PILP) mineralization process. Compared to traditional methods that produce only extrafibrillar hydroxyapatite (HA) clusters on the surface of collagen scaffolds, by using the PILP mineralization process, homogeneous intra- and extrafibrillar minerals were achieved on densified collagen films, leading to a similar nanostructure as bone, and a woven microstructure analogous to woven bone. The role of collagen cross-links on mineralization was examined and it was found that the cross-linked collagen films stimulated the mineralization reaction, which in turn enhanced the mechanical properties (hardness and modulus). The highest value of hardness and elastic modulus was 0.7 ± 0.1 and 9.1 ± 1.4 GPa in the dry state, respectively, which is comparable to that of woven bone. In the wet state, the values were much lower (177 ± 31 and 8 ± 3 MPa) due to inherent microporosity in the films, but still comparable to those of woven bone in the same conditions. Mineralization of collagen films with controllable mineral content and good mechanical properties provide a biomimetic route toward the development of bone substitutes for the next generation of biomaterials. This work also provides insight into understanding the role of collagen fibrils on mineralization.