A computational and experimental mechanical study of nanocomposites for 3D printed scaffolds with a new geometry.

We present a combined study of the mechanical properties of 3D printed scaffolds made by nanocomposite materials based on polycaprolactone (PCL). The geometry and dimensions of the three different systems is the same. Τhe porosity is 50% for all systems. Distributions of von-Mises strains and stresses, and total deformations were obtained through Finite Element Analysis (FEA) for a maximum amount of force applied, in a compressive numerical experiment. Also compressive experiments were performed for both raw and 3D nanoconposite scaffolds.

Human trichinellosis caused by Trichinella britovi in Greece, and literature review.

Trichinellosis is a cosmopolitan zoonotic parasitic disease caused by the nematodes of the genus Trichinella, through the consumption of raw or semi-raw infected meat from swine, horses and wild animals. This disease has been sporadically reported in Greece since 1946. The aim of the present study was to describe a trichinellosis case in a patient hospitalized in northern Greece, in 2017. A 47-year-old male was admitted to hospital with intense generalized myalgia, periorbital swelling, fever, exhaustion and anorexia. Biochemical and haematological profile showed eosinophilia and elevated creatine phosphokinase (CPK). Anti-Trichinella spp. IgG and IgM antibodies were detected by serology and Trichinella spp. larvae were found in two muscle biopsies by compressorium and histological examination. A larva collected from the muscle biopsy was identified as Trichinella britovi by polymerase chain reaction (PCR). Albendazole (400 mg twice per day × 10 days) was administered and the clinical condition of the patient promptly improved. This is the first identification of T. britovi in a patient in Greece.

Modeling of the interaction between osteoblasts and biocompatible substrates as a function of adhesion strength.

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. Nanoscale structured substrates [e.g., titania nanotubes (TNTs) or carbon nanotubes (CNTs)] dramatically improve the functions of conventional biomaterials. The present investigation evaluated the behavior of osteoblasts cells cultured on smooth and nanostructured substrates, by measuring osteoblasts specific biomarkers [alkaline phosphatase (AP) and total protein] and cells adhesion strength to substrates, followed by semi-empirical modeling to predict the experimental results. Findings were in total agreement with the current state of the art. The proliferation, as well as the AP and total protein levels were higher on the nanostructure phases (TNTs, CNTs) comparing to the smooth ones (plastic and pure titanium). Cells adhesion strength measured was found higher on the nanostructured materials. This coincided with a higher value of proteins which are directly implicated in the process of adherence. Results were accurately predicted through the Viscoelastic Hybrid Interphase Model. A gradual adherence of bone cells to implants using multilayered biomaterials that involve biodegradable polymeric films and a nanoscale modification of titanium surface is suggested to improve performance through an interphase-mediated osteointegration of orthopedic implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 621-628, 2018.

Acoustoelasticity in cancellous bone.

Strain-dependence of ultrasound speed in cancellous bone was determined by applying a range of uniaxial compressive strains in the elasticity region, in a single direction, parallel or perpendicular to the propagating wave. Compressive strain modulated the ultrasound speed significantly. The decrease of ultrasound speed was found to change linearly as a function of strain. The changes of broadband ultrasound attenuation were also determined for the two dilatational waves (parallel or perpendicular to the strain). They do not follow linear relation or constant sign of change with strain for the examined specimens. Considerable possibilities open up for using developments in acoustoelasticity for nondestructive ultrasonic techniques.

Towards a solution of the wires' slippage problem of the Ilizarov external fixator.

Clinical experience has indicated that many complications during treatment with the Ilizarov method, and mainly tract infection, are related to decreased wire tension. The aim of this work was to evaluate biomechanically a novel wire tensioning and clamping system that will minimise or even diminish the reduction of the wire pretension during treatment. The proposed approach is based on threading of the wire end in a sufficient length. The wire pretension is applied by twisting a nut on the threaded part of the wires against the ring and is recorded by an incorporated force sensor. For biomechanical evaluation, the frame, consisting of a polyethylene bar, simulating the bone fragment, suspended on two rings, was subjected to a dynamic load of 0-800 N at a frequency of 0.5 Hz. After dynamic loading for 20 min, loss of the initial wire pretension for the novel clamping system ranged between 12 and 16%. The average loss for conventionally clamped wires was 75%. The advantages of the novel clamping system were the much greater ability to sustain the transverse load and the easy and effectual wire re-tensioning. Although wire slippage has been avoided with the novel system, wire material yield is still responsible for a pretension loss.

Mechanoresponses of human primary osteoblasts grown on carbon nanotubes.

Bone mechanotransduction is strongly influenced by the biomaterial properties. A good understanding of these mechanosensory mechanisms in bone has the potential to provide new strategies in the highly evolving field of bone tissue engineering. The aim of the present investigation was to study the interactive effects of local mechanical stimuli on multiwalled carbon nanotubes (MWCNTs)/osteoblast interface, using an in vitro model that allows the study of cell growth, attachment and differentiation. Strain was applied at physiological levels [strain magnitudes 500 microstrain (µÉ›), at frequency of load application 0.5 Hz]. The effect of mechanical strain and substrate was thus studied by measuring the messenger RNA expression of alkaline phosphatase, vinculin, collagen 1A, and integrins ß1, ß3, α4, and αv, using real-time quantitative polymerase chain reaction. The osteoblasts grown on MWCNTs displayed quick adaptation to the new environment by modulating the expression of key adhesion integrins. Furthermore, the addition of mechanical strain interplayed with the extracellular matrix and was efficiently transduced by cells grown on MWCNTs, providing stronger adhesion and survival. MWCNTs are therefore a material perfectly compatible with osteoblast differentiation, adhesion, and growth, and should be further evaluated, to derive new-generation biomaterial scaffolds for the treatment of skeletal defects which require bone reconstruction.

Carbon nanotubes reinforced chitosan films: mechanical properties and cell response of a novel biomaterial for cardiovascular tissue engineering.

Carbon nanotubes have been proposed as fillers to reinforce polymeric biomaterials for the strengthening of their structural integrity to achieve better biomechanical properties. In this study, a new polymeric composite material was introduced by incorporating various low concentrations of multiwalled carbon nanotubes (MWCNTs) into chitosan (CS), aiming at achieving a novel composite biomaterial with superior mechanical and biological properties compared to neat CS, in order to be used in cardiovascular tissue engineering applications. Both mechanical and biological characteristics in contact with the two relevant cell types (endothelial cells and vascular myofibroblasts) were studied. Regarding the mechanical behavior of MWCNT reinforced CS (MWCNT/CS), 5 and 10 % concentrations of MWCNTs enhanced the mechanical behavior of CS, with that of 5 % exhibiting a superior mechanical strength compared to 10 % concentration and neat CS. Regarding biological properties, MWCNT/CS best supported proliferation of endothelial and myofibroblast cells, MWCNTs and MWCNT/CS caused no apoptosis and were not toxic of the examined cell types. Conclusively, the new material could be suitable for tissue engineering (TE) and particularly for cardiovascular TE applications.

Slow flow of passive neutrophils and sequestered nucleus into micropipette.

In the present study, the role of the nucleus and its contribution to the deformability of the passive neutrophils was investigated. To determine the rheological properties of the nucleus and of the neutrophil itself, deformation tests on single neutrophil and sequestered nucleus have been performed by micropipette under low aspiration pressure (80 Pa = 2-3 Pcr). The stiffness of the nucleus was found to be larger than that of the neutrophil, and its viscosity was found almost ten-fold higher. A subpopulation of neutrophils (Sub-A) showed two phases of deformation, a first rapid phase and a second phase with a constant deformation rate up to their full entrance, with an apparent viscosity mu app-second-Phase(N Sub-A) = 286 +/- 123 Pa x s, calculated by the liquid drop model. Another subpopulation (Sub-B) of the tested neutrophils displayed three deformation phases: a first one reflecting the rapid entry of cell into the micropipette, a second with constant deformation rate, and a third phase, with a slower, also constant, deformation rate were recorded. The corresponding apparent viscosities were found as mu app-second-Phase(N Sub-B) = 341 +/- 94 Pa x s and mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa x s. The apparent viscosity values of the neutrophilic nucleus, mu app (N nucl) = 2468 +/- 1345 Pa x s and of the whole neutrophil calculated in the third phase of deformation, mu app-third-Phase(N Sub-B) = 1651 +/- 734 Pa.s were comparable. These results support our hypothesis that the nucleus plays a significant role in the mechanical and rheological behavior of the neutrophil, especially when it has to pass through openings much smaller than its size.

Prediction of local cellular deformation in bone--influence of microstructure dimensions.

A multilevel finite element approach is applied to predict local cell deformations in bone tissue. The loads applied to a whole femur during the stance phase of the gait cycle were related to the strain at the level of a single lacuna and of canaliculi. Cell deformations were predicted from detailed linear FE analysis of the microstructure, consisting of an arrangement of cells embedded in bone matrix material. The local macroscopic stress assigned to each point was used to calculate the mesoscopic and consequently the microscopic nodal forces. The actual tissue principal strain along the femur deviated considerably from an average tissue value. The predicted bone matrix strains around osteocyte lacunae and canaliculi, were nonuniform and differed significantly from the macroscopically measured strains. Peak stresses and strains in the walls of the lacuna were up to six times those in the bulk extracellular matrix. Significant strain concentrations were observed at sites where the process meets the cell body. The dimensions of the lacunar axes influenced slightly the local strain.

Influence of microarchitecture alterations on ultrasonic backscattering in an experimental simulation of bovine cancellous bone aging.

An experimental model which can simulate physical changes that occur during aging was developed in order to evaluate the effects of change of mineral content and microstructure on ultrasonic properties of bovine cancellous bone. Timed immersion in hydrochloric acid was used to selectively alter the mineral content. Scanning electron microscopy and histological staining of the acid-treated trabeculae demonstrated a heterogeneous structure consisting of a mineralized core and a demineralized layer. The presence of organic matrix contributed very little to normalized broadband ultrasound attenuation (nBUA) and speed of sound. All three ultrasonic parameters, speed of sound, nBUA and backscatter coefficient, were sensitive to changes in apparent density of bovine cancellous bone. A two-component model utilizing a combination of two autocorrelation functions (a densely populated model and a spherical distribution) was used to approximate the backscatter coefficient. The predicted attenuation due to scattering constituted a significant part of the measured total attenuation (due to both scattering and absorption mechanisms) for bovine cancellous bone. Linear regression, performed between trabecular thickness values and estimated from the model correlation lengths, showed significant linear correlation, with R(2)=0.81 before and R(2)=0.80 after demineralization. The accuracy of estimation was found to increase with trabecular thickness.

Multilevel finite element modeling for the prediction of local cellular deformation in bone.

The underlying mechanisms by which bone cells respond to mechanical stimuli or how mechanical loads act on osteocytes housed in lacunae in bone are not well understood. In this study, a multilevel finite element (FE) approach is applied to predict local cell deformations in bone tissue. The local structure of the matrix dictates the local mechanical environment of an osteocyte. Cell deformations are predicted from detailed linear FE analysis of the microstructure, consisting of an arrangement of cells embedded in bone matrix material. This work has related the loads applied to a whole femur during the stance phase of the gait cycle to the strain of a single lacuna and of canaliculi. The predicted bone matrix strains around osteocyte lacunae and canaliculi were nonuniform and differed significantly from the macroscopically measured strains. Peak stresses and strains in the walls of the lacuna were up to six times those in the bulk extracellular matrix. Significant strain concentrations were observed at sites where the process meets the cell body.

Characterization of dense bovine cancellous bone tissue microstructure by ultrasonic backscattering using weak scattering models.

A weak scattering model was proposed for the ultrasonic frequency-dependent backscatter in dense bovine cancellous bone, using two autocorrelation functions to describe the medium: one with discrete homogeneities (spherical distribution of equal spheres) and another, which considers tissue as an inhomogeneous continuum (densely populated medium). The inverse problem to estimate trabecular thickness of bone tissue has been addressed. A combination of the two autocorrelation functions was required to closely approximate the backscatter from bovine bone with various microarchitecture, given that the shape of trabeculae ranges from a rodlike to a platelike shape. Because of the large variation in trabecular thickness, both at an intraspecimen and an interspecimen level, thickness distributions for individual trabeculae for each bone specimen were obtained, and dominant trabecular sizes were determined. Comparison of backscatter measurements to theoretical predictions indicated that there were more than one dominant trabecular sizes that scatter sound for most specimens. Linear regression, performed between dominant trabecular thickness and estimated correlation length, showed significant linear correlation (R(2)=0.81). Attenuation due to scattering by a continuous distribution of scatterers was predicted to be linear over a frequency range from 0.3 to 0.9 MHz, suggesting a possibility that scattering may be a significant source of attenuation.

Effect of surface roughness of the titanium alloy Ti-6Al-4V on human bone marrow cell response and on protein adsorption.

The effect of surface roughness of the titanium alloy Ti-6Al-4V (Ti alloy) on the short- and long-term response of human bone marrow cells in vitro and on protein adsorption was investigated. Three different values in a narrow range of surface roughness were used for the substrata (R(alpha): 0.320, 0.490 and 0.874 microm). Cell attachment, cell proliferation and differentiation (alkaline phosphatase specific activity) were determined past various incubation periods. The protein adsorption of bovine serum albumin and fibronectin, from single protein solutions, on rough and smooth Ti alloy surfaces was examined with two methods, X-ray photoelectron spectroscopy (XPS) and radiolabeling. Cell attachment and proliferation were surface roughness sensitive and increased as the roughness of Ti alloy increased. No statistically significant difference was observed in the expression of ALP activity on all three Ti alloy surfaces and culture plastic. Both methods, XPS and protein radiolabeling, showed that human serum albumin was adsorbed preferentially onto the smooth substratum. XPS technique showed that the rough substratum bound a higher amount of total protein (from culture medium supplied with 10% serum) and fibronectin (10-fold) than did the smooth one. The cell attachment may be explained by the differential adsorption of the two proteins onto smooth and rough Ti alloy surfaces.

Effectiveness of transfixation and length of instrumentation on titanium and stainless steel transpedicular spine implants.

This study compares the effectiveness of transfixation on the stiffness of two pedicle screw-rod constructs of different manufacture, implant design, and alloy, applied in one-and two-level instability. Four screws composed of either stainless steel or Titanium were assembled in pairs to two polymethylmethacrylate blocks to resemble one-and two-level corpectomy models and the construct underwent nondestructive torsional, extension, and flexion loading. In every loading test, each construct was tested using stainless steel or titanium rods of 4.9-mm diameter in two different lengths (short, 10 cm; long, 15 cm), not augmented or augmented with different transfixation devices or a pair of devices. The authors compared the stiffness of stainless steel and titanium constructs without cross-link with the stiffness of that reinforced with single or double Texas Scottish Rite Hospital (TSRH) cross-link, closed new-type cross-link (closed NTC), or open new-type cross-link (open NTC). The results showed that augmentation or no augmentation of short rods conferred significantly more stiffness than that of long rods of the same material in all three loading modes. The closed NTC provided the greatest increase of torsional, extension, and flexion stiffness, and single TSRH provided the least amount of stiffness. Torsional stiffness of short stainless steel rods augmented or not augmented was significantly greater than that of their titanium counterparts. Torsional stiffness of long titanium rods was always greater than that of their stainless steel counterparts. Extension stiffness of short nonaugmented titanium rods was superior to that of long titanium rods, whereas extension stiffness of nonaugmented short and long stainless steel rods was similar. Nonaugmented short titanium rods showed greater flexion stiffness than that of long titanium rods. Long stainless steel rods displayed significantly greater flexion stiffness than did their titanium counterparts. This nondestructive study showed that cross-links increase the torsional stiffness significantly but less so the flexion and extension stiffness of both titanium and stainless steel posterior transpedicular constructs. This increase was proportional to the cross-sectional diameter of the cross-link. Titanium constructs showed more torsional stiffness when used in two-level instability and steel showed more torsional stiffness in one-level instability, particularly when they are reinforced. Stainless steel constructs showed greater flexion stiffness when they were used in two-level and titanium showed greater flexion stiffness in one-level instability, particularly when they were reinforced with stiff cross-links. The effect of transfixation on extension forces was obvious when thick cross-links were used.

Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength.

Initial attachment of osteoblast cells and mineralization phenomena are generally enhanced on rough, sandblasted substrata. In the present work the effect of surface roughness of hydroxyapatite (HA) on human bone marrow cell response was investigated. Human bone marrow cells were plated onto HA disc-shaped pellets, prepared from synthetic HA powder. The pellets were sintered and polished with SiC paper 180-, 600- and 1200-grit, resulting in three surface roughness grades. Cell adhesion, proliferation and differentiation (evaluated with the expression of ALP activity) were determined following various incubation periods. Cell detachment strength was determined as the shear stress required to detach a given quantity of the adherent cells from the different substrata, using a rotating disc device that applied a linear range of shear stresses to the cells. The cells attached and grew faster on culture plastic in comparison with HA. No statistically significant differences were observed in the expression of ALP activity on all three HA surfaces and culture plastic. Cell adhesion, proliferation and detachment strength were surface roughness sensitive and increased as the roughness of HA increased. The percentage of the adherent cells decreased in a sigmoidal mode as a function of the applied shear stress. In conclusion, surface roughness of HA generally improved the short- and longer-term response of bone marrow cells in vitro. This behavior could be explained by the selective adsorption of serum proteins.

Adhesion strength of individual human bone marrow cells to fibronectin. Integrin beta1-mediated adhesion.

The purpose of this work was to study the adhesion strength of individual bone marrow cells, using a micropipette aspiration technique. The adhesion strength of the primary human bone marrow cells to fibronectin-coated substrate, by blocking the beta1 integrin with and without antibodies, was also determined. Human bone marrow stromal cells of the second passage were seeded at a density of 500 cells/cm2 on two different substrates: plastic culture dish (PCD) and PCD coated with fibronectin. In short adhesion times (15-180 min) the cells attached without spreading and remained almost spherical. A negative pressure of about 3500 Pa was applied, through the micropipette, on individual bone marrow cells and the detach process was recorded. The tip of the micropipette was bent at an 130 degrees angle to the corpus of the pipette and it was manipulated to be on the upper side of the cell and vertically to the bottom of the plate. It was observed from the experiments that the cells exhibited smaller adhesion strength at early adhesion times (30-85 min). After 85 min the adhesion strength increased abruptly and remained relatively constant for the adhesion period from 85 to 180 min for all substrates. Monoclonal antibodies against integrin subunit beta1 were used for integrin blocking experiments. The data suggested that the attachment of osteoblasts to a plastic culture dish without fibronectin coating occurred earlier than to the one coated with fibronectin PCD. In longer adhesion time the coating with fibronectin increased the adhesion strength at 107%. Blocking of integrin beta1 with monoclonal antibody resulted in decrease of the adhesion strength at 49%.

Factor analysis of the effectiveness of transfixation and rod characteristics on the TSRH screw-rod instrumentation.

The effects of Texas Scottish Rite Hospital (TSRH) hardware parameters (rod length and diameter and cross-link) and their interaction on the stiffness of the TSRH pedicle screw-rod construct were evaluated. Four TSRH screws were assembled in pairs to two polymethyl-methacrylate blocks to resemble a one-level or more corpectomy model and the construct underwent nondestructive torsional, extension, and flexion loading. In every loading test, each construct was tested using TSRH rods of different lengths (10, 15, and 20 cm) and diameters (4.9 and 6.5 mm) and different cross-links (TSRH and two new types made for this experiment). We compared the stiffness of the construct without cross-linking with that with single or double TSRH cross-linking, or either the closed new-type cross-link (closed NTC) or the open new-type cross-link (open NTC) using factor analysis. There was no axial slipping of one rod versus the other up to a force of 100 kg. The stiffness of the construct in all three loading modes increased as the rod length decreased, the rod diameter increased, and the construct was augmented with a cross-link. The closed NTC provided the greatest stiffness and the single TSRH provided the least stiffness. Unaugmented 10-cm-long rods showed two or three times more torsional stiffness than did that of the longer unaugmented rods independent of rod diameter. In addition, the closed NTC offered the maximal increase in flexion stiffness of the construct with thick rods and 10-, 15-, and 20-cm-long rods at a maximum of 40%, 27%, and 30%, respectively. This rigid closed NTC increased the extension stiffness of the same construct with 10- and 15-cm-long rods at 40% and 6%, respectively, whereas it had no influence on the extension stiffness of 20-cm-long rods.

Augmentation of anterior transvertebral screws using threaded teflon anchoring.

This is a biomechanical study to investigate the effect of augmentation of single anterior transvertebral screws. Two subsequent experiments (pullout and caudal loading) were performed in 78 porcine vertebral bodies of equal bone mineral density using 6.5-mm transvertebral screws augmented or not with specially designed Teflon anchoring. Three different types of 35- and 45-mm unaugmented screws (Kaneda, TSRH, and Zielke) were inserted at 90 degrees laterally in the vertebral body and were tested for pullout force. The pullout load to loosen the three different unaugmented screws did not significantly differ. The pullout force to loosen the 35- and 45-mm Zielke screws was 507 +/- 22 and 860 +/- 50 N, respectively. The corresponding pullout load to loosen the construct Zielke screw-Teflon anchoring was 1,005 +/- 148 N and 1,306 +/- 135 N for the 35- and 45-mm screws, respectively. When comparing the pullout force needed for unaugmented versus the augmented Zielke screw of the same length, there was a statistically significant (p < 0.0001) difference. During the caudal loading test, the unaugmented 45-mm Zielke screw showed that a uniform slope up to the yield point occurred at 0.35 +/- 0.12 mm of displacement, with an average tilting force for the tested screw of 1,362 +/- 151 N, when the screw became loose. The caudal loading test for the augmented Zielke screw was interrupted at 2,000 N because the load applied exceeded the load capacity of the testing machine, and thus no loosening occurred. The findings of this in vitro study showed that the Teflon anchoring provides superior anchorage and stability of single transvertebral Zielke screws. However, further biomechanical and clinical studies are required before using this device or its modification.

Test methodology for characterizing in vitro biodegradation.

In this paper mechanical tests for the characterization of the time-dependent behaviour of absorbable osteosynthetic materials are described. The tensile test is performed according to International Standard Organization (ISO) 3268/1978 and provides Young's modulus, tensile strength, elongation at tensile strength, and rupture force. The bending test is performed according to ISO 178/1975 and gives the flexural stress at various deflections, the force and deflection at break and the initial bending modulus. The torsional test is carried out according to ISO 458-1 and gives torsional load. The bending and torsional stress relaxation are measured. Relaxation modulus and the creep compliance are then determined. The parameters to be recorded in a cyclic bending test include the number of cycles or, in the case of sample survival, the remaining force at break at a bending test. The cyclic torsion test is carried out according to ISO 537/1989. The parameters to be recorded are the number of cycles or, in case of sample survival, the remaining force at a torsional test at 45 deg. Simulation tests provide a comparative assessment of the degradation phenomena under cyclic mechanical loading in bending or in torsion at elevated temperatures.

Stress relaxation behaviour of trabecular bone specimens.

The present study defines several conditions under which stress relaxation tests can be performed and investigates the viscoelastic behaviour of trabecular bone in compression through a series of stress relaxation tests at three strain levels and in three loading directions of each cubic specimen. A viscoelastic model is proposed to characterize the behaviour of trabecular bone and a spectrum of relaxation times is determined. Trabecular bone from the femoral head is non-linearly viscoelastic and displays anisotropic behaviour, which cannot be more symmetric elastically than orthotropic.