Wettability of mg-ha/Chitosan-based membrane surfaces: blood vs. autologous platelet liquid (APL).

OBJECTIVE: The physical and physical chemistry is able to influence the interaction of the scaffolds and bone substitutes with the body fluid and blood. The aim of the present investigation was to evaluate the wettability properties of an Mg-HA Chitosan-based Gel with blood vs. autologous platelet gel. MATERIALS AND METHODS: A total of 6 study groups were evaluated according to the Mg-HA Chitosan-based Gel thickness (1, 2 and 3 mm) and the fluids (blood vs. autologous platelet gel). The biomaterial wettability was conducted through the sessile drop technique. RESULTS: The study findings showed a significant difference in contact angle between the APL and blood groups (p<0.05). The MG-Ha Chitosan-based membrane thicknesses seem to produce no significant effects on contact angles measurement for all groups (p>0.05). CONCLUSIONS: In the present investigation, a similar MG/Ha gel membranes wettability was reported between APL and blood groups. In addition, a high hydrophilicity of MG/Ha gel membranes was reported with a potential advantage in terms of a more effective osteogenic capability in the clinical practice.

Ceramics with the signature of wood: a mechanical insight.

In an attempt to mimic the outstanding mechanical properties of wood and bone, a 3D heterogeneous chemistry approach has been used in a biomorphic transformation process (in which sintering is avoided) to fabricate ceramics from rattan wood, preserving its hierarchical fibrous microstructure. The resulting material (called biomorphic apatite â€‹[BA] henceforth) possesses a highly bioactive composition and is characterised by a multiscale hierarchical pore structure, based on nanotwinned hydroxyapatite lamellae, which is shown to display a lacunar fractal nature. The mechanical properties of BA are found to be exceptional (when compared with usual porous hydroxyapatite and other ceramics obtained from wood through sintering) and unique â€‹as they occupy a zone in the Ashby map previously free from ceramics, but not far from wood and bone. Mechanical tests show the following: (i) the strength in tension may exceed that in compression, (ii) failure in compression involves complex exfoliation patterns, thus resulting in high toughness, (iii) unlike in sintered porous hydroxyapatite, fracture does not occur 'instantaneously,' â€‹but its growth may be observed, and it exhibits tortuous patterns that follow the original fibrillar structure of wood, thus yielding outstanding toughness, (iv) the anisotropy of the elastic stiffness and strength show unprecedented values when situations of stresses parallel and orthogonal to the main channels are compared. Despite being a ceramic material, BA displays a mechanical behavior similar on the one hand to the ligneous material from which it was produced (therefore behaving as a 'ceramic with the signature of wood') and on the other hand to the cortical/spongy osseous complex constituting the structure of compact bone.

3D Patterning of cells in Magnetic Scaffolds for Tissue Engineering.

A three dimensional magnetic patterning of two cell types was realised in vitro inside an additive manufactured magnetic scaffold, as a conceptual precursor for the vascularised tissue. The realisation of separate arrangements of vascular and osteoprogenitor cells, labelled with biocompatible magnetic nanoparticles, was established on the opposite sides of the scaffold fibres under the effect of non-homogeneous magnetic gradients and loading magnetic configuration. The magnetisation of the scaffold amplified the guiding effects by an additional trapping of cells due to short range magnetic forces. The mathematical modelling confirmed the strong enhancement of the magnetic gradients and their particular geometrical distribution near the fibres, defining the preferential cell positioning on the micro-scale. The manipulation of cells inside suitably designed magnetic scaffolds represents a unique solution for the assembling of cellular constructs organised in biologically adequate arrangements.

Biomimetic hydroxyapatite/collagen composite drives bone niche recapitulation in a rabbit orthotopic model.

Synthetic osteoinductive materials that mimic the human osteogenic niche have emerged as ideal candidates to address this area of unmet clinical need. In this study, we evaluated the osteoinductive potential in a rabbit orthotopic model of a magnesium-doped hydroxyapatite/type I collagen â€‹(MHA/Coll) composite. The composite was fabricated to exhibit a highly fibrous structure of carbonated MHA with 70% (±2.1) porosity and a Ca/P ratio of 1.5 (±0.03) as well as a diverse range of elasticity separated to two distinct stiffness peaks of low (2.35 â€‹± â€‹1.16 â€‹MPa) and higher (9.52 â€‹± â€‹2.10 â€‹MPa) Young's Modulus. Data suggested that these specific compositional and nanomechanical material properties induced the deposition of de novo mineral phase, while modulating the expression of early and late osteogenic marker genes, in a 3D in vitro model using human bone marrow-derived mesenchymal stem cells (hBM-MSCs). When tested in the rabbit orthotopic model, MHA/Col1 scaffold induction of new trabecular bone mass was observed by DynaCT scan, only 2 weeks after implantation. Bone histomorphometry at 6 weeks revealed a significant amount of de novo bone matrix formation. qPCR demonstrated MHA/Coll scaffold full cellularization in vivo and the expression of both osteogenesis-associated genes (Spp1, Sparc, Col1a1, Runx2, Dlx5) as well as hematopoietic (Vcam1, Cd38, Sele, Kdr) and bone marrow stromal cell marker genes (Vim, Itgb1, Alcam). Altogether, these data provide â€‹evidence of the solid osteoinductive potential of MHA/Coll and its suitability for multiple approaches of bone regeneration.

Bone regeneration in a rabbit critical femoral defect by means of magnetic hydroxyapatite macroporous scaffolds.

Magnetic scaffolds have recently attracted significant attention in tissue engineering due to the prospect of improving bone tissue formation by conveying soluble factors such as growth factors, hormones, and polypeptides directly to the site of implantation, as well as to the possibility of improving implant fixation and stability. The objective of this study was to compare bone tissue formation in a preclinical rabbit model of critical femoral defect treated either with a hydroxyapatite (HA)/magnetite (90/10 wt %) or pure HA porous scaffolds at 4 and 12 weeks after implantation. The biocompatibility and osteogenic activity of the novel magnetic constructs was assessed with analysis of the amount of newly formed bone tissue and its nanomechanical properties. The osteoconductive properties of the pure HA were confirmed. The HA/magnetite scaffold was able to induce and support bone tissue formation at both experimental time points without adverse tissue reactions. Biomechanically, similar properties were obtained from nanoindentation analysis of bone formed following implantation of magnetic and control scaffolds. The results indicate that the osteoconductive properties of an HA scaffold are maintained following inclusion of a magnetic component. These provide a basis for future studies investigating the potential benefit in tissue engineering of applying magnetic stimuli to enhance bone formation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 546-554, 2018.

New hydroxyapatite nanophases with enhanced osteogenic and anti-bacterial activity.

This work describes the synthesis and characterization of new apatite phases co-doped with gallium, magnesium and carbonate, exhibiting osteogenic and antibacterial ability. The apatites are synthesized at low temperature to retain nanocrystallinity and controlled doping with the various bioactive foreign ions, as assessed by physico-chemical and crystallographic analyses, reporting the achievement of single phases with reduced crystal ordering. The analysis of single and multi-doped apatites reports to different mechanisms acting in the incorporation of gallium and magnesium ions in the apatite structure. The release of bioactive ions is correlated to the behavior of human mesenchymal stem cells and of different bacterial strands, selected among the most frequently affecting surgical procedures. Enhanced osteogenic and antibacterial ability is assessed in multi-doped apatites, thus suggesting potential future applications as new smart biomaterials integrating a significant boosting of bone regeneration with adequate protection against bacteria. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 106A: 521-530, 2018.

Needle-like ion-doped hydroxyapatite crystals influence osteogenic properties of PCL composite scaffolds.

Surface topography and chemistry both play a crucial role on influencing cell response in 3D porous scaffolds in terms of osteogenesis. Inorganic materials with peculiar morphology and chemical functionalities may be proficiently used to improve scaffold properties-in the bulk and along pore surface-promoting in vitro and in vivo osseous tissue in-growth. The present study is aimed at investigating how bone regenerative properties of composite scaffolds made of poly(Ɛ-caprolactone) (PCL) can be augmented by the peculiar properties of Mg(2+) ion doped hydroxyapatite (dHA) crystals, mainly emphasizing the role of crystal shape on cell activities mediated by microstructural properties. At the first stage, the study of mechanical response by crossing experimental compression tests and theoretical simulation via empirical models, allow recognizing a significant contribution of dHA shape factor on scaffold elastic moduli variation as a function of the relative volume fraction. Secondly, the peculiar needle-like shape of dHA crystals also influences microscopic (i.e. crystallinity, adhesion forces) and macroscopic (i.e. roughness) properties with relevant effects on biological response of the composite scaffold: differential scanning calorimetry (DSC) analyses clearly indicate a reduction of crystallization heat-from 66.75 to 43.05 J g(-1)-while atomic force microscopy (AFM) ones show a significant increase of roughness-from (78.15 ± 32.71) to (136.13 ± 63.21) nm-and of pull-off forces-from 33.7% to 48.7%. Accordingly, experimental studies with MG-63 osteoblast-like cells show a more efficient in vitro secretion of alkaline phosphatase (ALP) and collagen I and a more copious in vivo formation of new bone trabeculae, thus suggesting a relevant role of dHA to support the main mechanisms involved in bone regeneration.

Surface morphology, tribological properties and in vitro biocompatibility of nanostructured zirconia thin films.

Deposition of nanostructured and low-wear zirconia (ZrO2) thin films on the metallic component of a total joint implant is envisaged to reduce wear of the soft ultra-high molecular weight polyethylene (UHMWPE) counterpart. In this work, morphological surface features, wear resistance and in vitro-biocompatibility of zirconia thin films deposited by the novel Pulsed Plasma Deposition (PPD) method have been investigated. Film thickness, roughness and wettability were found to be strongly dependent on deposition gas pressure. Interestingly, wear rate of UHMWPE disks coupled to zirconia-coated titanium spheres was only poorly correlated to the contact angle values, while film roughness and thickness seemed not to affect it. Furthermore, wear of UHMWPE, when coupled with zirconia coated-titanium spheres, significantly decreased with respect to uncoated spheres under dry or NaCl-lubricated conditions; besides, when using bovine serum, similar results were obtained for coated and uncoated spheres. Finally, suitable mesenchymal stem and osteoblast cells adhesion, proliferation and viability were observed, suggesting good biocompatibility of the nanostructured zirconia films. Taken together, the results shown in this work indicate that zirconia thin films deposited by the PPD method deserve further investigations as low-wear materials for biomedical applications such as total joint replacement.

Mesenchymal stem cells and platelet gel improve bone deposition within CAD-CAM custom-made ceramic HA scaffolds for condyle substitution.

PURPOSE: This study evaluated the efficacy of a regenerative approach using mesenchymal stem cells (MSCs) and CAD-CAM customized pure and porous hydroxyapatite (HA) scaffolds to replace the temporomandibular joint (TMJ) condyle. METHODS: Pure HA scaffolds with a 70% total porosity volume were prototyped using CAD-CAM technology to replace the two temporomandibular condyles (left and right) of the same animal. MSCs were derived from the aspirated iliac crest bone marrow, and platelets were obtained from the venous blood of the sheep. Custom-made surgical guides were created by direct metal laser sintering and were used to export the virtual planning of the bone cut lines into the surgical environment. Sheep were sacrificed 4 months postoperatively. The HA scaffolds were explanted, histological specimens were prepared, and histomorphometric analysis was performed. RESULTS: Analysis of the porosity reduction for apposition of newly formed bone showed a statistically significant difference in bone formation between condyles loaded with MSC and condyles without (P < 0.05). The bone ingrowth (BI) relative values of split-mouth comparison (right versus left side) showed a significant difference between condyles with and without MSCs (P < 0.05). Analysis of the test and control sides in the same animal using a split-mouth study design was performed; the condyle with MSCs showed greater bone formation. CONCLUSION: The split-mouth design confirmed an increment of bone regeneration into the HA scaffold of up to 797% upon application of MSCs.

Modifying bone scaffold architecture in vivo with permanent magnets to facilitate fixation of magnetic scaffolds.

The fundamental elements of tissue regeneration are cells, biochemical signals and the three-dimensional microenvironment. In the described approach, biomineralized-collagen biomaterial functions as a scaffold and provides biochemical stimuli for tissue regeneration. In addition superparamagnetic nanoparticles were used to magnetize the biomaterials with direct nucleation on collagen fibres or impregnation techniques. Minimally invasive surgery was performed on 12 rabbits to implant cylindrical NdFeB magnets in close proximity to magnetic scaffolds within the lateral condyles of the distal femoral epiphyses. Under this static magnetic field we demonstrated, for the first time in vivo, that the ability to modify the scaffold architecture could influence tissue regeneration obtaining a well-ordered tissue. Moreover, the association between NdFeB magnet and magnetic scaffolds represents a potential technique to ensure scaffold fixation avoiding micromotion at the tissue/biomaterial interface.

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering.

In biomedicine, magnetic nanoparticles provide some attractive possibilities because they possess peculiar physical properties that permit their use in a wide range of applications. The concept of magnetic guidance basically spans from drug delivery and hyperthermia treatment of tumours, to tissue engineering, such as magneto-mechanical stimulation/activation of cell constructs and mechanosensitive ion channels, magnetic cell-seeding procedures, and controlled cell proliferation and differentiation. Accordingly, the aim of this study was to develop fully biodegradable and magnetic nanocomposite substrates for bone tissue engineering by embedding iron-doped hydroxyapatite (FeHA) nanoparticles in a poly(ε-caprolactone) (PCL) matrix. X-ray diffraction analyses enabled the demonstration that the phase composition and crystallinity of the magnetic FeHA were not affected by the process used to develop the nanocomposite substrates. The mechanical characterization performed through small punch tests has evidenced that inclusion of 10 per cent by weight of FeHA would represent an effective reinforcement. The inclusion of nanoparticles also improves the hydrophilicity of the substrates as evidenced by the lower values of water contact angle in comparison with those of neat PCL. The results from magnetic measurements confirmed the superparamagnetic character of the nanocomposite substrates, indicated by a very low coercive field, a saturation magnetization strictly proportional to the FeHA content and a strong history dependence in temperature sweeps. Regarding the biological performances, confocal laser scanning microscopy and AlamarBlue assay have provided qualitative and quantitative information on human mesenchymal stem cell adhesion and viability/proliferation, respectively, whereas the obtained ALP/DNA values have shown the ability of the nanocomposite substrates to support osteogenic differentiation.

Innovative magnetic scaffolds for orthopedic tissue engineering.

The use of magnetism in tissue engineering is a very promising approach, in fact magnetic scaffolds are able not only to support tissue regeneration, but they can be activated and work like a magnet attracting functionalized magnetic nanoparticles (MNPs) injected close to the scaffold enhancing tissue regeneration. This study aimed to assess the in vivo biocompatibility and osteointegrative properties of novel magnetic scaffolds. Two hydroxyapatite/collagen (70/30 wt %) magnetic scaffolds were magnetized with two different techniques: direct nucleation of biomimetic phase and superparamagnetic nanoparticles (MNPs) on self-assembling collagen fibers (MAG-A) and scaffold impregnation in ferro-fluid solution (MAG-B). Magnetic scaffolds were implanted in rabbit distal femoral epiphysis and tibial mid-diaphysis. Histopathological screening showed no inflammatory reaction due to MNPs. Significantly higher bone healing rate (ΔBHR) results were observed in MAG-A in comparison to MAG-B. Significant differences were also found between experimental times with an increase in ΔBHR from 2 to 4 weeks for both scaffolds in trabecular bone, while only for MAG-B (23%, p < 0.05) in cortical bone. The proposed magnetic scaffolds seem to be promising for magnetic guiding in orthopedic tissue engineering applications and they will be suitable to treat also several pathologies in regenerative medicine area.

Bioplasty for vertebral fractures: preliminary results of a pre-clinical study on goats using autologous modified skin fibroblasts.

The debate is still ongoing about the long term effects of the mininvasive vertebral augmentation techniques and their usefulness in treating more complex cases where a bone inducing effect more than a merely bone substitution would be suitable, such as the vertebral fractures in young patients. We previously developed a clinically relevant gene therapy approach using modified dermal fibroblasts for inducing bone healing and bone formation in different animal models. The aim of this study is to show the feasibility of a minimally invasive percutaneous intrasomatic ex vivo gene therapy approach to treat thoracolumbar vertebral fractures and anterior column bone defects in a goat model.

A conceptually new type of bio-hybrid scaffold for bone regeneration.

Magnetic bio-hybrid porous scaffolds have been synthesized, nucleating nano-apatite in situ on self-assembling collagen, in the presence of magnetite nano-particles. The magnetic phase acted as a sort of cross-linking agent for the collagen, inducing a chemico-physical-mechanical stabilization of the material and allowing us to control the porosity network of the scaffold. Gradients of bio-mineralization and magnetization were also developed for osteochondral application. The good potentiality of the material as a biomedical device, able to offer assistance to bone regeneration through scaffold reloading with specific factors guided by an external magnetic field, has been preliminarily investigated. Up to now the proof of this concept has been realized through in vitro assessments.

A novel route in bone tissue engineering: magnetic biomimetic scaffolds.

In recent years, interest in tissue engineering and its solutions has increased considerably. In particular, scaffolds have become fundamental tools in bone graft substitution and are used in combination with a variety of bio-agents. However, a long-standing problem in the use of these conventional scaffolds lies in the impossibility of re-loading the scaffold with the bio-agents after implantation. This work introduces the magnetic scaffold as a conceptually new solution. The magnetic scaffold is able, via magnetic driving, to attract and take up in vivo growth factors, stem cells or other bio-agents bound to magnetic particles. The authors succeeded in developing a simple and inexpensive technique able to transform standard commercial scaffolds made of hydroxyapatite and collagen in magnetic scaffolds. This innovative process involves dip-coating of the scaffolds in aqueous ferrofluids containing iron oxide nanoparticles coated with various biopolymers. After dip-coating, the nanoparticles are integrated into the structure of the scaffolds, providing the latter with magnetization values as high as 15 emu g(-)(1) at 10 kOe. These values are suitable for generating magnetic gradients, enabling magnetic guiding in the vicinity and inside the scaffold. The magnetic scaffolds do not suffer from any structural damage during the process, maintaining their specific porosity and shape. Moreover, they do not release magnetic particles under a constant flow of simulated body fluids over a period of 8 days. Finally, preliminary studies indicate the ability of the magnetic scaffolds to support adhesion and proliferation of human bone marrow stem cells in vitro. Hence, this new type of scaffold is a valuable candidate for tissue engineering applications, featuring a novel magnetic guiding option.

Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DmuCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

Accuracy of breath tests using low doses of 13C-urea to diagnose Helicobacter pylori infection: a randomised controlled trial.

BACKGROUND: The 13C-urea breath test (UBT) for detecting Helicobacter pylori infection is a non-invasive method based on the organism's urease activity. Since its first description, the method has been extensively modified. However, only the dose of 13C-urea and the measurement equipment are directly related to the cost of the test. AIMS: (1) To assess the diagnostic accuracy before eradication therapy of three UBTs using 25, 15, and 10 mg of 13C-urea, respectively; and (2) to determine diagnostic performance in the post-eradication setting showing the highest values for sensitivity and specificity with the lowest dose of 13C-urea. METHODS: Three hundred consecutive patients were randomised to be tested with one of the three UBTs. All patients underwent upper endoscopy with biopsies. A total of 222 more patients were enrolled to evaluate the second aim. Infected patients were offered treatment and asked to return 4-6 weeks after the end of therapy to perform endoscopic follow up and to carry out 13C-UBT. RESULTS: In the pretreatment setting, 13C-UBT 25 mg had a sensitivity of 100% (95% confidence interval (CI) 91.8-100) and a specificity of 100% (95% CI 93.7-100); 13C-UBT 15 mg had a sensitivity of 96.1% (95% CI 86.8-98.9) and a specificity of 100% (95% CI 92.6-100); and 13C-UBT 10 mg had a sensitivity of 89.1% (95% CI 77-95.3) and a specificity of 100% (95% CI 93.3-100). As the test with the best performance and the lowest dose of 13C-urea was 13C-UBT 15 mg, it was evaluated after treatment, reporting a sensitivity of 100% (95% CI 79.6-100) and a specificity of 98.9% (95% CI 94.3-99.8). DISCUSSION: UBTs using 25 and 15 mg of 13C-urea were both accurate in the diagnosis of H pylori infection in untreated patients. 13C-UBT 15 mg was also accurate for follow up of patients after treatment.

A 10-day levofloxacin-based triple therapy in patients who have failed two eradication courses.

BACKGROUND: A standard third-line treatment is lacking, and European guidelines recommend performing culture in these patients. However, the use of this procedure as 'routine practice' is definitively not feasible. AIM: To evaluate the eradication rate of a 10-day levofloxacin-based triple therapy in patients who have failed two eradication courses for Helicobacter pylori. METHODS: A total of 151 patients with persistent Helicobacter pylori infection after two treatments were studied. Patients were considered positive if two of three endoscopic tests were positive. Susceptibility testing was also performed. Patients received a standard dose of proton-pump inhibitors twice daily, levofloxacin 250 mg twice daily and amoxicillin 1 g twice daily, for 10 days. Endoscopic follow-up was carried out 4-6 weeks after the end of eradication therapy. RESULTS: About 76% (95% CI: 68.8-82.3), and 85% (95% CI: 77.5-89.7) of patients were eradicated according to intention-to-treat and per-protocol analysis, respectively. Eradication rates of the strains showed as 92% (95% CI: 83.2-96.7) of those resistant to both metronidazole and clarithromycin but susceptible to levofloxacin. CONCLUSIONS: In patients who failed previous regimens, the 10-day levofloxacin-based triple therapy is safe and effective, allowing eradication in almost 80% of the patients.

High-resolution 3D scaffold model for engineered tissue fabrication using a rapid prototyping technique.

Rapid prototyping, automatic image processing (computer-aided design (CAD)) and computer-aided manufacturing techniques are opening new and interesting prospects for medical devices and tissue engineering, especially for hard tissues such as bone. The development of a bone high-resolution scaffold prototype using these techniques is described. The results testify to the fidelity existing between microtomographic reconstruction and CAD. Furthermore, stereolithographic manufacturing of this scaffold, which possesses a high degree of similarity to the starting model as monitored by morphological evaluations (mean diameter 569 +/- 147 microm), represents a promising result for regenerative medicine applications.

From biomimetic apatites to biologically inspired composites.

Hydroxyapatite is an elective material for bone substitution. In this outline of our recent activity the crucial role of nanostructured ceramics in the design and preparation of ceramic scaffolds will be described, focussing on our more recent interest in biomimetic apatites, in particular apatites containing HPO42- CO32- and Mg2+ which are similar to the mineral component of bone. The paper describes such nanostructured products and, in particular, innovative synthetic techniques capable of yielding powders with higher reactivity and bioactivity. However, so far the characteristics of artificial bone tissues have been shown to be very different from those of natural bone, mainly because of the absence of the peculiar self-organizing interaction between apatites and the protein component. This causes modification of the structure of apatites and of the features of the overall composite forming human bone tissue. Therefore, attempts to mimic the features and structure of natural bone tissue, leading toward so-called bio-inspired materials, will be speculated upon. New techniques used to reproduce a composite in which a nanosize blade-like crystal of hydroxyapatite (HA) grows in contact with self-assembling fibres of natural polymer will be presented. In this specific case, the amazing ability of biological systems to store and process information at the molecular level, nucleating nanosize apatites (bio-inspired material), is exploited.