Despina Deligianni

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.

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.

Role of surface roughness of titanium versus hydroxyapatite on human bone marrow cells response.

This study was designed to assess human bone marrow cell response and particularly cell adhesion, proliferation, and differentiation, when cultured in vitro on titanium alloy and hydroxyapatite with different values of surface roughness. A further aim was to compare the cell response on the two materials, currently used in spinal surgery. Cell adhesion was determined after 0.5, 2, 4, and 18 hours of incubation; proliferation after 8, 11, 14, and 16 days of culture; and differentiation was evaluated with the expression of alkaline phosphatase activity after 8 and 16 days of culture. This study showed that bone marrow cells grew faster on titanium alloy than on hydroxyapatite, although fewer cells attached to titanium, compared to those attached to hydroxyapatite. No statistically significant difference was observed as the expression of alkaline phosphatase activity on hydroxyapatite and titanium alloy of the same roughness. Cell adhesion, proliferation, and differentiation are dependent on surface roughness of the biomaterial, and all three increased as the roughness of titanium alloy increased. Conclusively, surface roughness of titanium and hydroxyapatite significantly influences bone marrow cell response, and therefore these biomaterials should be used with rough outer surface, if a significant cell response on them is desired. These advantages of titanium and hydroxyapatite theoretically seem to be of particular importance in the following situations: long fusions, lumbosacral fusion, revision surgery with poor bone bank, neuropathic scoliosis associated with few bone graft reserves, and adult patients with severe osteoporosis.

Biomechanical evaluation of conventional internal contemporary spinal fixation techniques used for stabilization of complete sacroiliac joint separation: a 3-dimensional unilaterally isolated experimental stiffness study.

Study Design. Comparative 3-dimensional biomechanical testing. Objective. To compare 5 fixation techniques, 3 using screws or screw and plates and 2 spinal, used for stabilization of complete unilateral sacroiliac dislocation in composite models. Summary of Background Data. Harrington compression rods have been used for posterior iliosacral stabilization. Recently, the use of compact spinal instrumentation has been introduced for stabilization of iliosacral joint separation to achieve immediate and permanent stability, allowing early mobilization. To the authors’ knowledge, no comparative mechanical studies between commonly used internal fixation techniques and contemporary spinal instrumentation have been performed. Methods. Fifteen identical composite models of the left hemipelvis and sacrum were used to simulate consistently the “worst-case scenario” of complete unilateral sacroiliac dislocation. Subgroups of 3 models each were used to apply 5 (A–E) alternative fixation iliosacral joint fixation techniques: 1 multiaxial 7.5-mm Cotrel-Dubousset screw inserted in the posterior superior iliac spine and connected with a long Cotrel-Dubousset horizontal rod with 6.5 mm multiaxial Cotrel-Dubousset screws inserted bilaterally in the S1 pedicles (technique A); 1 multiaxial 7.5 mm Cotrel-Dubousset titanium pedicle screw inserted in the posterior superior iliac spine and connected with a short horizontal Cotrel-Dubousset-rod to a 6.5 mm multiaxial Cotrel-Dubousset-screw inserted to the ipsilateral S1 pedicle (technique B); 1, 6.5 mm cancellous AO-screw (technique C); 2, 6.5 mm cancellous AO screws (technique D); and 2 dynamic stainless steel compression plates (technique E) placed anteriorly. Constructs were biomechanically tested. The ilium was unilaterally rigidly fixed, the sacrum was put horizontal in the mediolateral direction with a forward tilt of 30° (close to physiologic conditions) in the sagittal plane, and a vertical quasi-static compressive load ranging from 0 to 500 N was applied on theendplate of S1, reproducing a “worst case” loading scenario. Construct stiffness, frontal plus sagittal kinematics, and iliosacral joint gap size for all 5 techniques were measured. Results. The construct stiffness (N/mm ± standard deviation) ranged for model: A, 121 ± 18; B, 78 ± 10; C, 168 ± 13; D, 193 ± 42; and E, 145 ± 4. All other parameters exhibited minor variations between the different techniques of fixation: at the 400 N load level, the maximum iliosacral gap globally ranged 0.9–2.8 mm, the maximum mediolateral sacral tilt ranged 1.3°–2.4°, and the maximum anteroposterior sacral tilt ranged 0.6°–3.0°. Conclusions. The iliosacral fixation with 2 6.5 mm AO-cancellous screws for complete sacroiliac dislocation demonstrated the highest stiffness and the short spinal instrumentation the poorest stiffness. All other fixation techniques could be generally considered of equivalent stability value.

Adhesion strength of individual human bone marrow cells to fibronectin. Integrin β1-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 β1 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° 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 β1 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 β1 with monoclonal antibody resulted in decrease of the adhesion strength at 49%.© 2001 Kluwer Academic Publishers

Effects of physiological mechanical strains on the release of growth factors and the expression of differentiation marker genes in human osteoblasts growing on Ti-6Al-4V

Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V/osteoblast interface, using an in vitro model. Homogeneous strain was applied to human bone marrow derived osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at physiological levels (strain magnitudes 500 microstrain (microepsilon) and 1000 microepsilon, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation.

Cellular Function and Adhesion Mechanisms of Human Bone Marrow Mesenchymal Stem Cells on Multi-walled Carbon Nanotubes

Multiwalled carbon nanotubes (MWCNTs) are considered to be excellent reinforcements for biorelated applications, but, before being incorporated into biomedical devices, their biocompatibility need to be investigated thoroughly. We investigated the ability of films of pristine MWCNTs to influence human mesenchymal stem cells’ proliferation, morphology, and differentiation into osteoblasts. Moreover, the selective integrin subunit expression and the adhesion mechanism to the substrate were evaluated on the basis of adherent cell number and adhesion strength, following the treatment of cells with blocking antibodies to a series of integrin subunits. Results indicated that MWCNTs accelerated cell differentiation to a higher extent than tissue culture plastic, even in the absence of additional biochemical inducing agents. The pre-treatment with anti-integrin antibodies decreased number of adherent cells and adhesion strength at 4–60%, depending on integrin subunit. These findings suggest that pristine MWCNTs represent a suitable reinforcement for bone tissue engineering scaffolds.

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.

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.

Estimation of hydrodynamic shear stresses developed on human osteoblasts cultured on Ti–6Al–4V and strained by four point bending. Effects of mechanical loading to specific gene expression

The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti–6Al–4V-osteoblast interface, using an in vitro model. Homogeneous strain was applied to Human Bone Marrow derived Osteoblasts (HBMDOs) cultured on Ti–6Al–4V, at levels which are considered physiological, by a four-point bending mechanostimulatory system. A simple model for the estimation of maximum hydrodynamic shear stresses developed on cell culture layer and induced by nutrient medium flow during mechanical loading, as a function of the geometry of the culture plate and the load characteristics, is proposed. Shear stresses were lower than those which can elicit cell response. Mechanical loading was found that contributes to the regulation of osteoblast differentiation by influencing the expression of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the osteocalcin expression, whereas osteopontin gene expression was unaffected by mechanical loading at all experimental conditions.