Poly(ε-caprolactone)/Hydroxyapatite 3D Honeycomb Scaffolds for a Cellular Microenvironment Adapted to Maxillofacial Bone Reconstruction.

The elaboration of biomimetic materials inspired from the specific structure of native bone is one the main goal of tissue engineering approaches. To offer the most appropriate environment for bone reconstruction, we combined electrospinning and electrospraying to elaborate an innovative scaffold composed of alternating layers of polycaprolactone (PCL) and hydroxyapatite (HA). In our approach, the electrospun PCL was shaped into a honeycomb-like structure with an inner diameter of 160 µm, capable of providing bone cells with a 3D environment while ensuring the material biomechanical strength. After 5 days of culture without any differentiation factor, the murine embryonic cell line demonstrated excellent cell viability on contact with the PCL-HA structures as well as active colonization of the scaffold. The cell differentiation, as tested by RT-qPCR, revealed a 6-fold increase in the expression of the RNA of the Bglap involved in bone mineralization as compared to a classical 2D culture. This differentiation of the cells into osteoblasts was confirmed by alkaline phosphatase staining of the scaffold cultivated with the cell lineage. Later on, organotypic cultures of embryonic bone tissues showed the high capacity of the PCL-HA honeycomb structure to guide the migration of differentiated bone cells throughout the cavities and the ridge of the biomaterial, with a colonization surface twice as big as that of the control. Taken together, our results indicate that PCL-HA honeycomb structures are biomimetic supports that promotes in vitro osteocompatibility, osteoconduction, and osteoinduction and could be suitable for being used for bone reconstruction in complex situations such as the repair of maxillofacial defects.

Electrospun honeycomb as nests for controlled osteoblast spatial organization.

Honeycomb nanofibrous scaffolds were elaborated by electrospinning onto micro-patterned collectors either with poly(ϵ-caprolactone) (PCL) or poly(D, L-lactic acid) (PLA). The unimodal distribution of fiber diameters, observed for PLA, led to relatively flat scaffolds; on the other hand, the bimodal distribution of PCL fiber diameters significantly increased the relief of the scaffolds' patterns due to the preferential deposition of the thick fiber portions on the walls of the collector's patterns via preferential electrostatic interaction. Finally, a biological evaluation demonstrated the effect of the scaffolds' relief on the spatial organization of MG63 osteoblast-like cells. Mimicking hemi-osteons, cell gathering was observed inside PCL honeycomb nests with a size ranging from 80 to 360 µm.

Production, structure and in vitro degradation of electrospun honeybee silk nanofibers.

Honeybees produce silken cocoons containing four related fibrous proteins. High levels of each of the honeybee silk proteins can be produced recombinantly by fermentation in Escherichia coli. In this study we have used electrospinning to fabricate a single recombinant honeybee silk protein, AmelF3, into nanofibers of around 200 nm diameter. Infrared spectroscopy found that the molecular structure of the nanofibers was predominantly coiled coil, essentially the same as native honeybee silk. Mats of the honeybee nanofibers were treated with methanol or by water annealing, which increased their ß-sheet content and rendered them water insensitive. The insoluble mats were degraded by protease on a time scale of hours to days. The protease gradually released proteins from the solid state and these were subsequently rapidly degraded into small peptides without the accumulation of partial degradation products. Cell culture assays demonstrated that the mats allowed survival, attachment and proliferation of fibroblasts. These results indicate that honeybee silk proteins meet many prerequisites for use as a biomaterial.

Multifunctionalized electrospun silk fibers promote axon regeneration in central nervous system.

The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.

Multilayer nanofilms as substrates for hepatocellular applications.

Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachment and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma (HepG2) cells, adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(l-lysine) (PLL) and poly(l-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after 7 days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)(n) (i.e. nPAH-PSS bilayers) and cross-linked (PLL-ALG)(n)-PLL. Cross-linked PLL-ALG and PLL-PGA films support attachment and function of ARH, independently of the terminal layer, provided collagen is adsorbed to the top of the film. (PAH-PSS)(n), cross-linked (PLL-ALG)(n), and cross-linked (PLL-PGA)(n)-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Fibronectin terminated multilayer films: protein adsorption and cell attachment studies.

Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)--a matrix protein known to promote cell adhesion--onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.

Probing adsorbed fibronectin layer structure by kinetic analysis of monoclonal antibody binding.

Adsorbed protein layers are often away from equilibrium and thus exhibit history dependent structures. We use the kinetics of monoclonal antibody binding, as measured using optical waveguide lightmode spectroscopy (OWLS), to investigate the structure of adsorbed fibronectin (Fn) layers formed under different kinetic paths. For all of the layers investigated, we find no difference between the apparent adsorption rate constants of (i) monoclonal antibodies specific to Fn's cell binding site (alpha-Fn) and (ii) monoclonal antibodies specific to cytochrome c (alpha-CC, as a control), indicating initial adsorption of antibodies to be non-specific. For certain layers, the saturation density and the initial projected area per antibody differ significantly between alpha-Fn and alpha-CC, suggesting specific binding to follow the initial non-specific attachment. The fraction of antibodies binding specifically to the Fn layer, and the number of Fn binding sites per specific binding event, are estimated in terms of the difference in initial projected areas between alpha-Fn and alpha-CC. For a Fn layer formed at a bulk concentration of 2 microg/mL, we find a decrease in specific binding with an increase in Fn layer formation time, suggesting post-adsorption structural changes of a lower density adsorbed layer diminish binding site availability. Conversely, for a Fn layer formed at a bulk concentration of 40 microg/mL, we find an increase in specific binding with an increase in the aging time of the Fn layer, implying post-adsorption structural changes reveal binding sites for a higher density adsorbed layer.