Osseointegration of polyethylene implants coated with titanium and biomimetically or electrochemically deposited hydroxyapatite in a rabbit model.

PURPOSE: The aim of this study was to evaluate the osseointegration of a new coating directly deposited on PE at room temperature. METHODS: Thirty-six (36) male New Zealand rabbits were randomly assigned to receive one out of three types of implants: two tested implants, i.e. PE implant coated with TiPVD and biomimetic HA (biomimetic), PE implant coated with TiPVD and electrolytic HA (electrolytic), and positive control made of massive microrough titanium coated with plasma sprayed HA (TiHAPS). Osseointegration was evaluated by histomorphometry (bone tissue in contact [BIC]), mineralized bone area [MBA]) and mechanical testing (push-out test, interfacial shear strength [ISS]) at six and 12 weeks in the distal femurs. RESULTS: For BIC there were no differences between the groups at six (p = 0.98) and 12 weeks (p = 0.13). For MBA, no statistically significant difference was measured between groups at six (p = 0.52) and 12 weeks (p = 0.57). At six weeks, interfacial shear strength (ISS) was significantly higher (p = 0.01) for TiHAPs implants compared to biomimetic and electrolytic implants. This difference was not significant at 12 weeks (p = 0.92). CONCLUSION: The osseointegration of biomimetic and electrolytic implants was equivalent to a positive control at 12 weeks.

Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor.

Background: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. Methods: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. Results: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. Conclusion: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Mechanisms of pore formation in hydrogel scaffolds textured by freeze-drying.

Whereas freeze-drying is a widely used method to produce porous hydrogel scaffolds, the mechanisms of pore formation involved in this process remained poorly characterized. To explore this, we focused on a cross-linked polysaccharide-based hydrogel developed for bone tissue engineering. Scaffolds were first swollen in 0.025% NaCl then freeze-dried at low cooling rate, i.e. -0.1 °C min-1, and finally swollen in aqueous solvents of increasing ionic strength. We found that scaffold's porous structure is strongly conditioned by the nucleation of ice. Electron cryo-microscopy of frozen scaffolds demonstrates that each pore results from the growth of one to a few ice grains. Most crystals were formed by secondary nucleation since very few nucleating sites were initially present in each scaffold (0.1 nuclei cm-3 °C-1). The polymer chains are rejected in the intergranular space and form a macro-network. Its characteristic length scale coincides with the ice grain size (160 µm) and is several orders of magnitude greater than the mesh size (90 nm) of the cross-linked network. After sublimation, the ice grains are replaced by macro-pores of 280 µm mean size and the resulting dry structure is highly porous, i.e. 93%, as measured by high-resolution X-ray tomography. In the swollen state, the scaffold mean pore size decreases in aqueous solvent of increasing ionic strength (120 µm in 0.025% NaCl and 54 µm in DBPS) but the porosity remains the same, i.e. 29% regardless of the solvent. Finally, cell seeding of dried scaffolds demonstrates that the pores are adequately interconnected to allow homogenous cell distribution. STATEMENT OF SIGNIFICANCE: The fabrication of hydrogel scaffolds is an important research area in tissue engineering. Hydrogels are textured to provide a 3D-framework that is favorable for cell proliferation and/or differentiation. Optimum hydrogel pore size depends on its biological application. Producing porous hydrogels is commonly achieved through freeze-drying. However, the mechanisms of pore formation remain to be fully understood. We carefully analyzed scaffolds of a cross-linked polysaccharide-based hydrogel developed for bone tissue engineering, using state-of-the-art microscopic techniques. Our experimental results evidenced the shaping of hydrogel during the freezing step, through a specific ice-templating mechanism. These findings will guide the strategies for controlling the porous structure of hydrogel scaffolds.

Mesoscale porosity at the dentin-enamel junction could affect the biomechanical properties of teeth.

In this paper, the 3D-morphology of the porosity in dentin is investigated within the first 350µm from the dentin-enamel junction (DEJ) by fluorescence confocal laser scanning microscopy (CLSM). We found that the porous microstructure exhibits a much more complex geometry than classically described, which may impact our fundamental understanding of the mechanical behavior of teeth and could have practical consequences for dental surgery. Our 3D observations reveal numerous fine branches stemming from the tubules which may play a role in cellular communication or mechanosensing during the early stages of dentinogenesis. The effect of this highly branched microstructure on the local mechanical properties is investigated by means of numerical simulations. Under simplified assumptions on the surrounding tissue characteristics, we find that the presence of fine branches negatively affects the mechanical properties by creating local stress concentrations. However, this effect is reduced by the presence of peritubular dentin surrounding the tubules. The porosity was also quantified using the CSLM data and compared to this derived from SEM imaging. A bimodal distribution of channel diameters was found near the DEJ with a mean value of 1.5-2µm for the tubules and 0.3-0.5µm for the fine branches which contribute to 30% of the total porosity (∼1.2%). A gradient in the branching density was observed from the DEJ towards the pulp, independently of the anatomical location. Our work constitutes an incentive towards more elaborate multiscale studies of dentin microstructure to better assess the effect of aging and for the design of biomaterials used in dentistry, e.g. to ensure more efficient bonding to dentin. Finally, our analysis of the tubular network structure provides valuable data to improve current numerical models.

The inhibition of cell spreading on a cellulose substrate (cuprophan) induces an apoptotic process via a mitochondria-dependent pathway.

Cell shape was found to be a strong indicator of whether individual cells grow or die, and may play an important role in controlling apoptosis as well as cell growth. We compared here the behaviour of rounded Swiss 3T3 cells aggregated on a cellulose cuprophan membrane to those cultured on dish polystyrene. We demonstrated that cells aggregated on cellulose substrates for up to 48 h underwent programmed cell death that was associated with phosphatidylserine flipping and caspase 9 and caspase 3 activation, suggesting a mitochondria-dependent apoptotic process. In addition, we found that this phenomenon cannot be entirely explained by disengagement of alpha 5 beta 1 integrin ligation.